Anti-TIGIT antigen-binding proteins and methods of use thereof

ABSTRACT

Provided herein are antigen-binding proteins (ABPs) that selectively bind to TIGIT and its isoforms and homologs, and compositions comprising the ABPs. Also provided are methods of using the ABPs, such as therapeutic and diagnostic methods.

This application is a divisional of U.S. application Ser. No.15/430,998, filed Feb. 13, 2017, which is a continuation ofPCT/US2016/054484, filed Sep. 29, 2016, which claims the benefit of U.S.Provisional Application No. 62/235,990, filed Oct. 1, 2015, each ofwhich is hereby incorporated by reference in its entirety, for allpurposes.

FIELD

Provided herein are antigen-binding proteins (ABPs) with bindingspecificity for T cell immunoreceptor with Ig and ITIM domains (TIGIT)and compositions comprising such ABPs, including pharmaceuticalcompositions, diagnostic compositions, and kits. Also provided aremethods of making TIGIT ABPs, and methods of using TIGIT ABPs, forexample, for therapeutic purposes, diagnostic purposes, and researchpurposes.

BACKGROUND

TIGIT has been identified as a co-inhibitory receptor that limits theresponse of T cells to cancer and chronic infection. See Grogan et al.,J. Immunol., 2014, 192: (1 Supplement) 203.15, incorporated by referencein its entirety. Blockade of TIGIT has been shown to contribute to theenhancement CD8+ T cell effector function, and improvement of viralclearance and tumor rejection. See id.

Thus, there is a need for therapeutics that can antagonize TIGIT.Provided herein are ABPs that fulfill this need.

SUMMARY

Provided herein are ABPs that specifically bind TIGIT and methods ofusing such ABPs.

In some embodiments, the TIGIT is selected from human TIGIT (“hTIGIT”,SEQ ID NO:1), cynomolgus monkey TIGIT (“cTIGIT”, SEQ ID NO:2), andmurine TIGIT (“mTIGIT”, SEQ ID NO:3 or 138).

In some embodiments, the ABP comprises an antibody. In some aspects, theantibody is a monoclonal antibody. In some aspects, the antibody is achimeric antibody. In some aspects, the antibody is a humanizedantibody. In some aspects, the antibody is a human antibody. In someaspects, the ABP comprises an antibody fragment. In some embodiments,the ABP comprises an alternative scaffold.

In some embodiments, the TIGIT is expressed on the surface of a targetcell. In some aspects, the ABP antagonizes TIGIT expressed on thesurface of the target cell.

In some embodiments, the target cell is selected from an effector Tcell, a regulatory T cell, a natural killer (NK) cell, and a naturalkiller T (NKT) cell. In some aspects, the target cell is an effector Tcell selected from a helper (CD4-positive, “CD4+”) T cell, a cytotoxic(CD8-positive, “CD8+”) T cell, and combinations thereof. In someaspects, the target cell is a regulatory T cell selected from aCD4+CD25+Foxp3+ regulatory T cell, a CD8+CD25+ regulatory T cell, andcombinations thereof.

In some embodiments, the ABPs provided herein induce various biologicaleffects associated with inhibition of TIGIT. In some aspects, an ABPprovided herein prevents inhibition of an effector T cell. In someaspects, the ABP co-stimulates an effector T cell. In some aspects, theABP inhibits the suppression of an effector T cell by a regulatory Tcell. In some aspects, the ABP increases the number of effector T cellsin a tissue or in systemic circulation. In some aspects, the tissue is atumor. In some aspects, the tissue is a tissue that is infected with avirus.

Also provided are kits comprising one or more of the ABPs providedherein, and instructions for use of the ABPs. Also provided are kitscomprising one or more of the pharmaceutical compositions providedherein, and instructions for use of the pharmaceutical composition.

Also provided are isolated polynucleotides encoding the ABPs providedherein, and portions thereof.

Also provided are vectors comprising such polynucleotides.

Also provided are recombinant host cells comprising such polynucleotidesand recombinant host cells comprising such vectors.

Also provided are methods of producing an ABP provided herein using thepolynucleotides, vectors, or host cells provided herein.

Also provided are pharmaceutical compositions comprising the ABPsprovided herein and a pharmaceutically acceptable excipient.

Also provided are methods of treating or preventing a disease orcondition in a subject in need thereof, comprising administering to thesubject an effective amount of an ABP provided herein, or apharmaceutical composition comprising such ABP. In some aspects, thedisease or condition is a cancer. In some aspects, the disease orcondition is a viral infection. In some aspects the method furthercomprises administering one or more additional therapeutic agents. Insome aspects, the additional therapeutic agent is an immunostimulatoryagent.

In some embodiments, provided herein is a first family of ABPs, whereinan ABP of such family comprises the following six CDR sequences: (a) aCDR-H3 having the sequence A-R-D-G-V-L-X₁-L-N-K-R-S-F-D-I, wherein X₁ isA or T (SEQ ID NO: 128); (b) a CDR-H2 having the sequenceS-I-Y-Y-S-G-X₂-T-Y-Y-N-P-S-L-K-S, wherein X₂ is S, Q or G (SEQ ID NO:129); (c) a CDR-H1 having the sequence G-S-I-X₃-S-G-X₄-Y-Y-W-G, whereinX₃ is E or A, and X₄ is L, V or S (SEQ ID NO: 130); (d) a CDR-L3 havingthe sequence QQHTVRPPLT (SEQ ID NO: 64); (e) a CDR-L2 having thesequence GASSRAT (SEQ ID NO: 68); and (f) a CDR-L1 having the sequenceRASQSVSSSYLA (SEQ ID NO: 71). In some embodiments, provided herein is anABP within such first family.

In some embodiments, an ABP of such first family comprises: (a) a CDR-H3of SEQ ID NO: 32, a CDR-H2 of SEQ ID NO: 40, a CDR-H1 of SEQ ID NO: 54,a CDR-L3 of SEQ ID NO: 64, a CDR-L2 of SEQ ID NO: 68, and a CDR-L1 ofSEQ ID NO: 71; (b) a CDR-H3 of SEQ ID NO: 31, a CDR-H2 of SEQ ID NO: 40,a CDR-H1 of SEQ ID NO: 54, a CDR-L3 of SEQ ID NO: 64, a CDR-L2 of SEQ IDNO: 68, and a CDR-L1 of SEQ ID NO: 71; (c) a CDR-H3 of SEQ ID NO: 31, aCDR-H2 of SEQ ID NO: 39, a CDR-H1 of SEQ ID NO: 51, a CDR-L3 of SEQ IDNO: 64, a CDR-L2 of SEQ ID NO: 68, and a CDR-L1 of SEQ ID NO: 71; (d) aCDR-H3 of SEQ ID NO: 31, a CDR-H2 of SEQ ID NO: 40, a CDR-H1 of SEQ IDNO: 52, a CDR-L3 of SEQ ID NO: 64, a CDR-L2 of SEQ ID NO: 68, and aCDR-L1 of SEQ ID NO: 71; or (e) a CDR-H3 of SEQ ID NO: 31, a CDR-H2 ofSEQ ID NO: 41, a CDR-H1 of SEQ ID NO: 53, a CDR-L3 of SEQ ID NO: 64, aCDR-L2 of SEQ ID NO: 68, and a CDR-L1 of SEQ ID NO: 71.

In some embodiments, an ABP of such first family comprises: (a) a V_(H)sequence of SEQ ID NO: 13 and a V_(L) sequence of SEQ ID NO: 26; (b) aV_(H) sequence of SEQ ID NO: 12 and a V_(L) sequence of SEQ ID NO: 26;(c) a V_(H) sequence of SEQ ID NO: 14 and a V_(L) sequence of SEQ ID NO:26; (d) a V_(H) sequence of SEQ ID NO: 15 and a V_(L) sequence of SEQ IDNO: 26; (e) a V_(H) sequence of SEQ ID NO: 9 and a V_(L) sequence of SEQID NO: 26; (f) a V_(H) sequence of SEQ ID NO: 10 and a V_(L) sequence ofSEQ ID NO: 26; or (g) a V_(H) sequence of SEQ ID NO: 11 and a V_(L)sequence of SEQ ID NO: 26.

In some embodiments, an ABP of such first family comprises: (a) (i) aheavy chain of SEQ ID NO: 99 and a light chain of SEQ ID NO: 92; or (ii)a heavy chain of SEQ ID NO: 100 and a light chain of SEQ ID NO: 92; (b)(i) a heavy chain of SEQ ID NO: 97 and a light chain of SEQ ID NO: 92;or (ii) a heavy chain of SEQ ID NO: 98 and a light chain of SEQ ID NO:92; (c) (i) a heavy chain of SEQ ID NO: 101 and a light chain of SEQ IDNO: 92; or (ii) a heavy chain of SEQ ID NO: 102 and a light chain of SEQID NO: 92; (d) (i) a heavy chain of SEQ ID NO: 103 and a light chain ofSEQ ID NO: 92; or (ii) a heavy chain of SEQ ID NO: 104 and a light chainof SEQ ID NO: 92; (e) (i) a heavy chain of SEQ ID NO: 90 and a lightchain of SEQ ID NO: 92; or (ii) a heavy chain of SEQ ID NO: 91 and alight chain of SEQ ID NO: 92; (f) (i) a heavy chain of SEQ ID NO: 93 anda light chain of SEQ ID NO: 92; or (ii) a heavy chain of SEQ ID NO: 94and a light chain of SEQ ID NO: 92; or (g) (i) a heavy chain of SEQ IDNO: 95 and a light chain of SEQ ID NO: 92; or (ii) a heavy chain of SEQID NO: 96 and a light chain of SEQ ID NO: 92.

In some embodiments, provided herein is a second family of ABPs, whereinan ABP of such family comprises the following six CDR sequences: (a) aCDR-H3 having the sequence A-R-D-A-N-Y-Y-G-X₁-A-W-A-F-D-P, wherein X₁ isS or G (SEQ ID NO: 131); (b) a CDR-H2 having the sequenceS-I-Y-Y-S-G-X₂-T-F-Y-N-P-S-L-K-X₃, wherein X₂ is S or A, and X₃ is S orG (SEQ ID NO: 132); (c) a CDR-H1 having the sequenceG-S-I-X₄-S-X₅-X₆-X₇-Y-W-G, wherein X₄ is S or T, X₅ is S or T, X₆ is Sor K, and X₇ is H or Y (SEQ ID NO: 133); (d) a CDR-L3 having thesequence QQHFNLPT (SEQ ID NO: 63); (e) a CDR-L2 having the sequenceDASNRAT (SEQ ID NO: 67); and (f) a CDR-L1 having the sequenceRASQSVSSYLA (SEQ ID NO: 70). In some embodiments, provided herein is anABP within such second family.

In some embodiments, an ABP of such second family comprises: (a) aCDR-H3 of SEQ ID NO: 29, a CDR-H2 of SEQ ID NO: 37, a CDR-H1 of SEQ IDNO: 49, a CDR-L3 of SEQ ID NO: 63, a CDR-L2 of SEQ ID NO: 67, and aCDR-L1 of SEQ ID NO: 70; (b) a CDR-H3 of SEQ ID NO: 30, a CDR-H2 of SEQID NO: 37, a CDR-H1 of SEQ ID NO: 50, a CDR-L3 of SEQ ID NO: 63, aCDR-L2 of SEQ ID NO: 67, and a CDR-L1 of SEQ ID NO: 70; (c) a CDR-H3 ofSEQ ID NO: 29, a CDR-H2 of SEQ ID NO: 38, a CDR-H1 of SEQ ID NO: 50, aCDR-L3 of SEQ ID NO: 63, a CDR-L2 of SEQ ID NO: 67, and a CDR-L1 of SEQID NO: 70; (d) a CDR-H3 of SEQ ID NO: 29, a CDR-H2 of SEQ ID NO: 36, aCDR-H1 of SEQ ID NO: 48, a CDR-L3 of SEQ ID NO: 63, a CDR-L2 of SEQ IDNO: 67, and a CDR-L1 of SEQ ID NO: 70; or (e) a CDR-H3 of SEQ ID NO: 29,a CDR-H2 of SEQ ID NO: 37, a CDR-H1 of SEQ ID NO: 50, a CDR-L3 of SEQ IDNO: 63, a CDR-L2 of SEQ ID NO: 67, and a CDR-L1 of SEQ ID NO: 70.

In some embodiments, an ABP of such second family comprises: (a) a V_(H)sequence of SEQ ID NO: 5 and a V_(L) sequence of SEQ ID NO: 25; (b) aV_(H) sequence of SEQ ID NO: 7 and a V_(L) sequence of SEQ ID NO: 25;(c) a V_(H) sequence of SEQ ID NO: 8 and a V_(L) sequence of SEQ ID NO:25; (d) a V_(H) sequence of SEQ ID NO: 4 and a V_(L) sequence of SEQ IDNO: 25; or (e) a V_(H) sequence of SEQ ID NO: 6 and a V_(L) sequence ofSEQ ID NO: 25.

In some embodiments, an ABP of such second family comprises: (a) (i) aheavy chain of SEQ ID NO: 82 and a light chain of SEQ ID NO: 81; or (ii)a heavy chain of SEQ ID NO: 83 and a light chain of SEQ ID NO: 81; (b)(i) a heavy chain of SEQ ID NO: 86 and a light chain of SEQ ID NO: 81;or (ii) a heavy chain of SEQ ID NO: 87 and a light chain of SEQ ID NO:81; (c) (i) a heavy chain of SEQ ID NO: 88 and a light chain of SEQ IDNO: 81; or (ii) a heavy chain of SEQ ID NO: 89 and a light chain of SEQID NO: 81; (d) (i) a heavy chain of SEQ ID NO: 79 and a light chain ofSEQ ID NO: 81; or (ii) a heavy chain of SEQ ID NO: 80 and a light chainof SEQ ID NO: 81; or (e) (i) a heavy chain of SEQ ID NO: 84 and a lightchain of SEQ ID NO: 81; or (ii) a heavy chain of SEQ ID NO: 85 and alight chain of SEQ ID NO: 81.

In some embodiments, provided herein is a third family of ABPs, whereinan ABP of such family comprises the following six CDR sequences: (a) aCDR-H3 having the sequence A-R-G-G-R-T-T-W-I-G-A-X₁-D-I, wherein X₁ is For L (SEQ ID NO: 134); (b) a CDR-H2 having the sequenceI-I-N-P-S-X₂-G-L-T-S-Y-A-X₃-K-F-Q-G, wherein X₂ is L or I, and X₃ is Qor R (SEQ ID NO: 135); (c) a CDR-H1 having the sequenceY-T-F-X₄-X₅-Y-Y-X₆-H, wherein X₄ is G, P or R, X₅ is N, A or E, and X₆is M or I (SEQ ID NO: 136); (d) a CDR-L3 having the sequence QQYVVWPPLT(SEQ ID NO:65); (e) a CDR-L2 having the sequence GASTRAT (SEQ ID NO:69);and (f) a CDR-L1 having the sequence RASQSVSSNLA (SEQ ID NO:72). In someembodiments, provided herein is an ABP within such third family.

In some embodiments, an ABP of such third family comprises: (a) a CDR-H3of SEQ ID NO: 33, a CDR-H2 of SEQ ID NO: 43, a CDR-H1 of SEQ ID NO: 60,a CDR-L3 of SEQ ID NO: 65, a CDR-L2 of SEQ ID NO: 69, and a CDR-L1 ofSEQ ID NO: 72; (b) a CDR-H3 of SEQ ID NO: 34, a CDR-H2 of SEQ ID NO: 43,a CDR-H1 of SEQ ID NO: 60, a CDR-L3 of SEQ ID NO: 65, a CDR-L2 of SEQ IDNO: 69, and a CDR-L1 of SEQ ID NO: 72; (c) a CDR-H3 of SEQ ID NO: 33, aCDR-H2 of SEQ ID NO: 44, a CDR-H1 of SEQ ID NO: 59, a CDR-L3 of SEQ IDNO: 65, a CDR-L2 of SEQ ID NO: 69, and a CDR-L1 of SEQ ID NO: 72; (d) aCDR-H3 of SEQ ID NO: 33, a CDR-H2 of SEQ ID NO: 42, a CDR-H1 of SEQ IDNO: 58, a CDR-L3 of SEQ ID NO: 65, a CDR-L2 of SEQ ID NO: 69, and aCDR-L1 of SEQ ID NO: 72; (e) a CDR-H3 of SEQ ID NO: 33, a CDR-H2 of SEQID NO: 42, a CDR-H1 of SEQ ID NO: 59, a CDR-L3 of SEQ ID NO: 65, aCDR-L2 of SEQ ID NO: 69, and a CDR-L1 of SEQ ID NO: 72; or (f) a CDR-H3of SEQ ID NO: 34, a CDR-H2 of SEQ ID NO: 44, a CDR-H1 of SEQ ID NO: 61,a CDR-L3 of SEQ ID NO: 65, a CDR-L2 of SEQ ID NO: 69, and a CDR-L1 ofSEQ ID NO: 72.

In some embodiments, an ABP of such third family comprises: (a) a V_(H)sequence of SEQ ID NO: 18 and a V_(L) sequence of SEQ ID NO: 27; (b) aV_(H) sequence of SEQ ID NO: 19 and a V_(L) sequence of SEQ ID NO: 27;(c) a V_(H) sequence of SEQ ID NO: 21 and a V_(L) sequence of SEQ ID NO:27; (d) a V_(H) sequence of SEQ ID NO: 16 and a V_(L) sequence of SEQ IDNO: 27; (e) a V_(H) sequence of SEQ ID NO: 17 and a V_(L) sequence ofSEQ ID NO: 27; or (f) a V_(H) sequence of SEQ ID NO: 20 and a V_(L)sequence of SEQ ID NO: 27.

In some embodiments, an ABP of such third family comprises: (a) (i) aheavy chain of SEQ ID NO: 110 and a light chain of SEQ ID NO: 107; or(ii) a heavy chain of SEQ ID NO: 111 and a light chain of SEQ ID NO:107; (b) (i) a heavy chain of SEQ ID NO: 112 and a light chain of SEQ IDNO: 107; or (ii) a heavy chain of SEQ ID NO: 113 and a light chain ofSEQ ID NO: 107; (c) (i) a heavy chain of SEQ ID NO: 116 and a lightchain of SEQ ID NO: 107; or (ii) a heavy chain of SEQ ID NO: 117 and alight chain of SEQ ID NO: 107; (d) (i) a heavy chain of SEQ ID NO: 105and a light chain of SEQ ID NO: 107; or (ii) a heavy chain of SEQ ID NO:106 and a light chain of SEQ ID NO: 107; (e) (i) a heavy chain of SEQ IDNO: 108 and a light chain of SEQ ID NO: 107; or (ii) a heavy chain ofSEQ ID NO: 109 and a light chain of SEQ ID NO: 107; or (f) (i) a heavychain of SEQ ID NO: 114 and a light chain of SEQ ID NO: 107; or (ii) aheavy chain of SEQ ID NO: 115 and a light chain of SEQ ID NO: 107.

In some embodiments, provided herein is a fourth family of ABPs, whereinan ABP of such family comprises the following six CDR sequences: (a) aCDR-H3 having the sequence ARLHVSGSYYPAYLDY (SEQ ID NO: 35); (b) aCDR-H2 having the sequence X₁-I-N-P-S-M-G-A-T-S-Y-X₂-Q-K-F-X₃-G, whereinX₁ is V or I, X₂ is A or T, and X₃ is Q or R (SEQ ID NO: 137); (c) aCDR-H1 having the sequence YTFTSHYMG (SEQ ID NO: 62); (d) a CDR-L3having the sequence QQYIVFPWT (SEQ ID NO: 66); (e) a CDR-L2 having thesequence GASTRAT (SEQ ID NO: 69); and (f) a CDR-L1 having the sequenceRASQSVSSNLA, (SEQ ID NO: 72). In some embodiments, provided herein is anABP within such fourth family.

In some embodiments, an ABP of such fourth family comprises: (a) aCDR-H3 of SEQ ID NO: 35, a CDR-H2 of SEQ ID NO: 46, a CDR-H1 of SEQ IDNO: 62, a CDR-L3 of SEQ ID NO: 66, a CDR-L2 of SEQ ID NO: 69, and aCDR-L1 of SEQ ID NO: 72; (b) a CDR-H3 of SEQ ID NO: 35, a CDR-H2 of SEQID NO: 47, a CDR-H1 of SEQ ID NO: 62, a CDR-L3 of SEQ ID NO: 66, aCDR-L2 of SEQ ID NO: 69, and a CDR-L1 of SEQ ID NO: 72; or (c) a CDR-H3of SEQ ID NO: 35, a CDR-H2 of SEQ ID NO: 45, a CDR-H1 of SEQ ID NO: 62,a CDR-L3 of SEQ ID NO: 66, a CDR-L2 of SEQ ID NO: 69, and a CDR-L1 ofSEQ ID NO: 72.

In some embodiments, an ABP of such fourth family comprises: (a) a V_(H)sequence of SEQ ID NO: 23 and a V_(L) sequence of SEQ ID NO: 28; (b) aV_(H) sequence of SEQ ID NO: 24 and a V_(L) sequence of SEQ ID NO: 28;or (c) a V_(H) sequence of SEQ ID NO: 22 and a V_(L) sequence of SEQ IDNO: 28.

In some embodiments, an ABP of such fourth family comprises: (a) (i) aheavy chain of SEQ ID NO: 121 and a light chain of SEQ ID NO: 120; or(ii) a heavy chain of SEQ ID NO: 122 and a light chain of SEQ ID NO:120; (b) (i) a heavy chain of SEQ ID NO: 123 and a light chain of SEQ IDNO: 120; or (ii) a heavy chain of SEQ ID NO: 124 and a light chain ofSEQ ID NO: 120; or (c) (i) a heavy chain of SEQ ID NO: 118 and a lightchain of SEQ ID NO: 120; or (ii) a heavy chain of SEQ ID NO: 119 and alight chain of SEQ ID NO: 120.

In some embodiments, provided herein is an isolated antigen bindingprotein (ABP) that specifically binds human TIGIT (hTIGIT; SEQ ID NO:1), comprising: (a) a CDR-H3 having at least about 80% identity to aCDR-H3 of a V_(H) region selected from SEQ ID NOs: 4-24; (b) a CDR-H2having at least about 80% identity to a CDR-H2 of a V_(H) regionselected from SEQ ID NOs: 4-24; (c) a CDR-H1 having at least about 80%identity to a CDR-H1 of a V_(H) region selected from SEQ ID NOs: 4-24;(d) a CDR-L3 having at least about 80% identity to a CDR-L3 of a V_(L)region selected from SEQ ID NOs: 25-28; (e) a CDR-L2 having at leastabout 80% identity to a CDR-L2 of a V_(L) region selected from SEQ IDNOs: 25-28; and (f) a CDR-L1 having at least about 80% identity to aCDR-L1 of a V_(L) region selected from SEQ ID NOs: 25-28. In someembodiments, the CDR-H3, CDR-H2, CDR-H1, CDR-L3, CDR-L2, and CDR-L1 areeach identified according to a numbering scheme selected from the Kabatnumbering scheme, the Chothia numbering scheme, or the IMGT numberingscheme. In some embodiments, the CDR-H1 is identified as defined by boththe Chothia and Kabat numbering schemes, inclusive of the boundaries ofboth numbering schemes. In some embodiments: (a) the CDR-H3 comprises aCDR-H3 selected from SEQ ID NOs: 29-35, or a variant thereof having 1,2, or 3 amino acid substitutions; (b) the CDR-H2 comprises a CDR-H3selected from SEQ ID NOs: 36-47, or a variant thereof having 1, 2, or 3amino acid substitutions; (c) the CDR-H1 comprises a CDR-H1 selectedfrom SEQ ID NOs: 48-54 or 58-62, or a variant thereof having 1 or 2amino acid substitutions; (d) the CDR-L3 comprises a CDR-L3 selectedfrom SEQ ID NOs: 63-66, or a variant thereof having 1 or 2 amino acidsubstitutions; (e) the CDR-L2 comprises a CDR-L2 selected from SEQ IDNOs: 67-69, or a variant thereof having 1 amino acid substitution; and(f) the CDR-L1 comprises a CDR-L1 selected from SEQ ID NOs: 70-72, or avariant thereof having 1 or 2 amino acid substitutions.

In some embodiments, provided herein is an isolated antigen bindingprotein (ABP) that specifically binds human TIGIT (hTIGIT; SEQ ID NO:1), comprising: (a) a V_(H) region having at least about 90% identity toa V_(H) region selected from SEQ ID NOs: 4-24; and (b) a V_(L) regionhaving at least about 90% identity to a V_(L) region selected from SEQID NOs: 25-28. In some embodiments: (a) the a V_(H) region comprises aV_(H) region selected from SEQ ID NOs: 4-24, or a variant thereof having1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions; and(b) the a V_(L) region comprises a V_(L) region selected from SEQ IDNOs: 25-28, or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions. In some embodiments, the amino acidsubstitutions are conservative amino acid substitutions.

In some embodiments, any of the foregoing ABPs: (a) competes for bindingto TIGIT with an antibody selected from MAB1, MAB2, MAB3, MAB4, MAB5,MAB6, MAB7, MAB8, MAB9, MAB10, MAB11, MAB12, MAB13, MAB14, MAB15, MAB16,MAB17, MAB18, MAB19, MAB20, or MAB21, each as provided in Table 5 ofthis disclosure; (b) inhibits binding of CD155 to TIGIT; (c) inhibitsbinding of CD112 to TIGIT; (d) inhibits association of CD226 with TIGIT;(e) activates an effector T cell or a natural killer (NK) cell; (f)decreases the number of regulatory T cells in a tissue or incirculation; (g) inhibits the suppression of an effector T cell by aregulatory T cell; (h) does not bind specifically to any of PVRL1,PVRL2, PVRL3, or PVRL4; or (i) is capable of any combination of (a)-(h).

In some embodiments, any of the foregoing ABPs: (a) specifically bindscynomolgus monkey TIGIT (cTIGIT; SEQ ID NO: 2); (b) binds murine TIGIT(mTIGIT; SEQ ID NO: 3) with an affinity lower (as indicated by higherKD) than the affinity of the ABP for hTIGIT, or does not bind mTIGIT; or(c) is capable of any combination of (a)-(b).

In some embodiments, any of the foregoing ABPs: (a) specifically bindscTIGIT (SEQ ID NO: 2); (b) binds mTIGIT (SEQ ID NO: 3) with an affinitylower (as indicated by higher K_(D)) than the affinity of the ABP forhTIGIT and cTIGIT; and (c) inhibits binding of CD155 to TIGIT.

In some embodiments, provided herein is an ABP that competes for bindingto TIGIT with any of the foregoing ABPs, wherein the ABP: (a)specifically binds cTIGIT (SEQ ID NO: 2); (b) binds mTIGIT (SEQ ID NO:3) with an affinity lower (as indicated by higher K_(D)) than theaffinity of the ABP for hTIGIT and cTIGIT; and (c) inhibits binding ofCD155 to TIGIT.

In some embodiments, any of the foregoing ABPs comprises an antibody. Insome embodiments, the antibody is a monoclonal antibody. In someembodiments, the antibody is selected from a human antibody, a humanizedantibody or a chimeric antibody.

In some embodiments, any of the foregoing ABPs is multispecific. In someembodiments, the multispecific ABP binds more than one antigen (i.e.,TIGIT and a different (non-TIGIT) antigen). In some embodiments, themultispecific ABP binds more than one epitope on a single antigen (i.e.,two or more epitopes on TIGIT).

In some embodiments, any of the foregoing ABPs comprises an antibodyfragment.

In some embodiments, any of the foregoing ABPs comprises an alternativescaffold.

In some embodiments, any of the foregoing ABPs comprises animmunoglobulin constant region. In some embodiments, the ABP comprisesheavy chain constant region of a class selected from IgA, IgD, IgE, IgG,or IgM. In some embodiments, the ABP comprises a heavy chain constantregion of the class IgG and a subclass selected from IgG4, IgG1, IgG2,or IgG3.

In some embodiments, any of the foregoing ABPs binds hTIGIT (SEQ IDNO: 1) with a K_(D) of less than about 10 nM, as measured by biolayerinterferometry. In some embodiments, any of the foregoing ABPs bindshTIGIT (SEQ ID NO: 1) with a K_(D) of less than about 5 nM, as measuredby biolayer interferometry. In some embodiments, any of the foregoingABPs binds hTIGIT (SEQ ID NO: 1) with a K_(D) of less than about 2 nM,as measured by biolayer interferometry. In some embodiments, any of theforegoing ABPs binds cTIGIT (SEQ ID NO: 2) with a K_(D) of less thanabout 100 nM, as measured by biolayer interferometry. In someembodiments, any of the foregoing ABPs binds cTIGIT (SEQ ID NO: 2) witha K_(D) of less than about 10 nM, as measured by biolayerinterferometry. In some embodiments, any of the foregoing ABPs shows nosignificant binding to mTIGIT in a biolayer interferometry assay. Insome embodiments, any of the foregoing ABPs binds to cell surface mTIGITwith a K_(D) of less than about 50 nM. In some embodiments, mTIGITcomprises SEQ ID NO: 3. In some embodiments, mTIGIT comprises SEQ ID NO:138.

In some embodiments, any of the foregoing ABPs comprises a polypeptidesequence having a pyroglutamate (pE) residue at its N-terminus. In someembodiments, any of the foregoing ABPs comprises a V_(H) sequence inwhich an N-terminal Q is substituted with pE. In some embodiments, anyof the foregoing ABPs comprises a V_(L) sequence in which an N-terminalE is substituted with pE. In some embodiments, any of the foregoing ABPscomprises a heavy chain sequence in which an N-terminal Q is substitutedwith pE. In some embodiments, any of the foregoing ABPs comprises alight chain sequence in which an N-terminal E is substituted with pE.

In some embodiments, provided herein are any of the foregoing ABPs foruse as a medicament. In some embodiments, provided herein are any of theforegoing ABPs for use in the treatment of a cancer or viral infection.In some embodiments, provided herein are any of the foregoing ABPs foruse in the treatment of a cancer, wherein the cancer is selected from asolid tumor and a hematological tumor. In some embodiments, providedherein are any of the foregoing ABPs for use as a medicament in thetreatment of a disease or condition that was not responsive to a priortherapy. In some embodiments, the prior therapy was a therapy comprisingan agent that inhibits the interaction between PD-1 and PD-L1.

In some embodiments, provided herein is an isolated polynucleotideencoding any of the foregoing ABPs, a V_(H) thereof, a V_(L) thereof, alight chain thereof, a heavy chain thereof or an antigen-binding portionthereof. In some embodiments, provided herein is a vector comprising thepolynucleotide. In some embodiments, provided herein is a host cellcomprising the polynucleotide and/or the vector. In some embodiments,provided herein is a method of producing any of the foregoing ABPs,comprising expressing the ABP in the host cell and isolating theexpressed ABP.

In some embodiments, provided herein is a pharmaceutical compositioncomprising any of the foregoing ABPs. In some embodiments, the amount ofthe ABP in the pharmaceutical composition is sufficient to (a) increaseeffector T cell activity; (b) increase cytolytic T cell activity; (c)increase NK cell activity; (d) inhibit TIGIT-mediated signaling; (e)inhibit or block the binding of CD155 and or CD112 to TIGIT; or (f) anycombination of (a)-(e), in a subject. In some embodiments, the any ofthe foregoing pharmaceutical compositions further comprises an antibodythat antagonizes PD-1 or blocks PD-L1 from interacting with PD-1. Insome embodiments, any of the foregoing pharmaceutical compositions isfor use as a medicament. In some embodiments, any of the foregoingpharmaceutical compositions is for use in the treatment of a cancer or aviral infection. In some embodiments, any of the foregoingpharmaceutical compositions is for use in the treatment of a cancer,wherein the cancer is selected from a solid tumor and a hematologicaltumor. In some embodiments, any of the foregoing pharmaceuticalcompositions is for use as a medicament in the treatment of a disease orcondition that was not responsive to a prior therapy. In someembodiments, the prior therapy was a therapy comprising an agent thatinhibits the interaction between PD-1 and PD-L1.

In some embodiments, provided herein is a method of treating orpreventing a disease or condition in a subject in need thereof,comprising administering to the subject an effective amount of any ofthe foregoing ABPs or any of the foregoing pharmaceutical compositions.In some embodiments, the disease or condition is a cancer or viralinfection. In some embodiments, the disease or condition is a cancer,and the cancer is selected from a solid tumor and a hematological tumor.In some embodiments, provided herein is a method of modulating an immuneresponse in a subject in need thereof, comprising administering to thesubject an effective amount of any of the foregoing ABPs or any of theforegoing pharmaceutical compositions. In some embodiments, any of theforegoing methods, further comprise administering one or more additionaltherapeutic agents to the subject. In some embodiments, the additionaltherapeutic agent is selected from an agent that inhibits theinteraction between PD-1 and PD-L1, a chemotherapy, an immunostimulatoryagent, radiation, and combinations thereof.

In some embodiments, the additional therapeutic agent for use in any ofthe foregoing methods of treatment, uses of an ABP or uses of apharmaceutical composition is an agent that inhibits the interactionbetween PD-1 and PD-L1, and wherein the agent that inhibits theinteraction between PD-1 and PD-L1 is selected from an antibody, apeptidomimetic, a small molecule or a nucleic acid encoding such agent.In some embodiments, the agent that inhibits the interaction betweenPD-1 and PD-L1 is selected from is selected from pembrolizumab,nivolumab, atezolizumab, avelumab, durvalumab, BMS-936559,sulfamonomethoxine 1, and sulfamethizole 2.

In some embodiments, the additional therapeutic agent for use in any ofthe foregoing methods of treatment, uses of an ABP or uses of apharmaceutical composition is an immunostimulatory agent selected from(a) an agent that blocks signaling of an inhibitory receptor of animmune cell or a ligand thereof or a nucleic acid encoding such agent;(b) an agonist to a stimulatory receptor of an immune cell or a nucleicacid encoding such agonist; (c) a cytokine or a nucleic acid encoding acytokine; (d) an oncolytic virus or a nucleic acid encoding an oncolyticvirus; (e) a T cell expressing a chimeric antigen receptor; (f) a bi- ormulti-specific T cell directed antibody or a nucleic acid encoding suchantibody; (g) an anti-TGF-β antibody or a nucleic acid encoding suchantibody; (h) a TGF-β trap or a nucleic acid encoding such trap; (i) avaccine to a cancer-associated antigen, including such antigen or anucleic acid encoding such antigen and (j) combinations thereof. In someembodiments, the additional therapeutic agent is an agent that blockssignaling of an inhibitory receptor of an immune cell or a ligandthereof or a nucleic acid encoding such agent, and the inhibitoryreceptor or ligand thereof is selected from CTLA-4, PD-1, PD-L1, PD-L2,LAG-3, Tim3, neuritin, BTLA, CECAM-1, CECAM-5, VISTA, LAIR1, CD160, 2B4,TGF-R, KIR, and combinations thereof. In some embodiments, theadditional therapeutic agent is an agonist to a stimulatory receptor ofan immune cell or a nucleic acid encoding such agonist, and thestimulatory receptor of an immune cell is selected from OX40, CD2, CD27,CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR,CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160,B7-H3, CD83 ligand, and combinations thereof. In some embodiments, theadditional therapeutic agent is a cytokine or a nucleic acid encoding acytokine selected from IL-2, IL-5, IL-7, IL-12, IL-15, IL-21, andcombinations thereof. In some embodiments, the additional therapeuticagent is an oncolytic virus or a nucleic acid encoding an oncolyticvirus selected from herpes simplex virus, vesicular stomatitis virus,adenovirus, Newcastle disease virus, vaccinia virus, a maraba virus, andcombinations thereof.

In some embodiments, the additional therapeutic agent is formulated inthe same pharmaceutical composition as the ABP. In some embodiments, theadditional therapeutic agent is formulated in a different pharmaceuticalcomposition from the ABP. In some embodiments, the additionaltherapeutic agent is administered prior to administering the ABP. Insome embodiments, the additional therapeutic agent is administered afteradministering the ABP. In some embodiments, the additional therapeuticagent is administered contemporaneously with the ABP.

In some embodiments, the subject is a subject that has been treated withan agent that inhibits the interaction between PD-1 and PD-L1 prior toperforming such method.

In some embodiments, the disease or condition afflicting the subject wasnot responsive to a prior therapy. In some embodiments, the priortherapy was a therapy comprising an agent that inhibits the interactionbetween PD-1 and PD-L1.

In some embodiments, provided herein is a kit comprising any of theforegoing pharmaceutical compositions, and instructions for the use ofsuch pharmaceutical composition. In some embodiments, the kit furthercomprises an additional pharmaceutical composition comprising anadditional therapeutic agent and instructions for the use of suchadditional therapeutic agent. In some embodiments, the additionaltherapeutic agent is selected from an agent that inhibits theinteraction between PD-1 and PD-L1, a chemotherapy, an immunostimulatoryagent, radiation, and combinations thereof. In some embodiments, theadditional therapeutic agent is an agent that inhibits the interactionbetween PD-1 and PD-L1, and wherein the agent inhibits the interactionbetween PD-1 and PD-L1 is selected from an antibody, a peptidomimetic, asmall molecule or a nucleic acid encoding such agent. In someembodiments, the agent that inhibits the interaction between PD-1 andPD-L1 is selected from is selected from pembrolizumab, nivolumab,atezolizumab, avelumab, durvalumab, BMS-936559, sulfamonomethoxine 1,and sulfamethizole 2. In some embodiments, the additional therapeuticagent is an immunostimulatory agent selected from (a) an agent thatblocks signaling of an inhibitory receptor of an immune cell or a ligandthereof or a nucleic acid encoding such agent; (b) an agonist to astimulatory receptor of an immune cell or a nucleic acid encoding suchagonist; (c) a cytokine or a nucleic acid encoding a cytokine; (d) anoncolytic virus or a nucleic acid encoding an oncolytic virus; (e) a Tcell expressing a chimeric antigen receptor; (f) a bi- or multi-specificT cell directed antibody or a nucleic acid encoding such antibody; (g)an anti-TGF-β antibody or a nucleic acid encoding such antibody; (h) aTGF-β trap or a nucleic acid encoding such trap; (i) a vaccine to acancer-associated antigen, including such antigen or a nucleic acidencoding such antigen and (j) combinations thereof. In some embodiments,the additional therapeutic agent is an agent that blocks signaling of aninhibitory receptor of an immune cell or a ligand thereof or a nucleicacid encoding such agent, and the inhibitory receptor or ligand thereofis selected from CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, Tim3, neuritin,BTLA, CECAM-1, CECAM-5, VISTA, LAIR1, CD160, 2B4, TGF-R, KIR, andcombinations thereof. In some embodiments, the additional therapeuticagent is an agonist to a stimulatory receptor of an immune cell or anucleic acid encoding such agonist, and the stimulatory receptor of animmune cell is selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40,BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83ligand, and combinations thereof. In some embodiments, the additionaltherapeutic agent is a cytokine or a nucleic acid encoding a cytokineselected from IL-2, IL-5, IL-7, IL-12, IL-15, IL-21, and combinationsthereof. In some embodiments, the additional therapeutic agent is anoncolytic virus or a nucleic acid encoding an oncolytic virus selectedfrom herpes simplex virus, vesicular stomatitis virus, adenovirus,Newcastle disease virus, vaccinia virus, a maraba virus, andcombinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show sequence alignments of various molecules describedfurther in the Examples.

FIG. 1A shows sequence alignments of the TIGIT extracellular domainsfrom human (SEQ ID NO: 139), cynomolgus monkey (SEQ ID NO: 140), mouse(residues 17-139 of SEQ ID NO: 3), and rat (SEQ ID NO: 141) TIGITreference sequences. FIG. 1B shows an alignment of the human TIGIT (SEQID NO: 142) and PVRL4 (SEQ ID NO: 139) extracellular domains.

FIG. 2 is an illustration of the similarity between the CD28-CTLA4 andCD226-TIGIT pathways and thus the utility of TIGIT as an immunecheckpoint target. The CD226/TIGIT costimulation/coinhibition biology isanalogous to that of CD28/CTLA4; TIGIT provides an inhibitory signal toT cells while CD226 provides a costimulation signal to T cells. TIGITligands CD155 and CD112 are widely expressed in tumors providing animmune suppressive environment.

FIG. 3 shows a schematic diagram of the TIGIT Jurkat/anti-CD3 HT-1080co-culture assay described in Example 7.

FIG. 4A shows the EC₅₀ curves from exemplary experiments comparing theability of MAB10 and an IgG4 isotype control to induce IL-2 productionin engineered Jurkat cells expressing human TIGIT. FIG. 4B shows theEC₅₀ curves from exemplary experiments comparing the ability of MAB10and an IgG4 isotype control to induce IL-2 production in engineeredJurkat cells expressing cynomolgus monkey TIGIT.

FIG. 5A-5C show TIGIT expression analysis on human CD4+ T cells by FACS.FIG. 5A and FIG. 5B show a series of graphs showing expression analysisof TIGIT, PVR and CD226 on unstimulated (FIG. 5A) and stimulated (FIG.5B) CD4+ T cells. FIG. 5C shows analysis of TIGIT expression on naïve(positive for CD45RA, a marker of naïve T cells, top right panel) andmemory (positive for CD45RO, a marker of activated T cells, bottom rightpanel) CD4+ T cells.

FIGS. 6A-6O show the effect of treatment with MABs on sub-optimallystimulated PBMCs from human donors. MABs were tested for their abilityto induce IFN-γ in PBMCs from human donors, including a control IgG4antibody (FIG. 6A), MAB2 (FIG. 6B), MAB3 (FIG. 6C), MAB4 (FIG. 6D), MAB5(FIG. 6E), MAB10 (FIG. 6F), MAB11 (FIG. 6G), MAB12 (FIG. 6H), MAB15(FIG. 6I), MAB16 (FIG. 6J), and SEC1 (hamster anti-mouse TIGIT, FIG.6K). Treatment of the PBMCs from Donor 1 with MAB10 induces theupregulation of several pro-inflammatory cytokines, including tumornecrosis factor alpha (TNF, FIG. 6L), lymphotoxin alpha (LT-α, FIG. 6M),and interferon gamma (IFN-γ, FIG. 6N). FIG. 6O shows a graphillustrating the EC₅₀ of MAB10 in PBMCs from Donor 1, as measured byIFN-γ production.

FIGS. 7A-7E provide a series of graphs showing the effect of MAB10 oncytokine secretion in sub-optimally stimulated PBMCs from Donor 2,including IFN-γ (FIG. 7A), TNF (FIG. 7B), interleukin 6 (IL-6, FIG. 7C),granulocyte macrophage colony-stimulating factor (GM-CSF, FIG. 7D), andLT-α(FIG. 7E). Data from cells treated with MAB10 are shown as blackbars and data from cells treated with the IgG4 isotype control are shownas light gray bars. Antibody concentration for each bar is in μg/mL.

FIGS. 8A-8C provide a series of graphs showing that the antagonistanti-TIGIT antibody MAB10 increases IFN-γ in a CD4+ cell assay usingcells obtained from three different donors. FIG. 8A shows resultsobtained from CD4+ cells obtained from Donor 1. FIG. 8B shows resultsobtained from CD4+ cells obtained from Donor 2. FIG. 8C shows resultsobtained from CD4+ cells obtained from Donor 3. IFN-γ production incells treated with either MAB10 (black bars) or the IgG4 isotype control(light gray bars) is shown in the left panel of each of FIGS. 8A-8C. Theaverage EC₅₀ value for MAB10 in this assay was calculated by determiningthe concentration of MAB10 required to induce 50% of the increase inIFN-γ signal (plotted in the right panel of each of FIGS. 8A-8C).

FIG. 9A shows the results of the assay in which a 1:1 ratio of MAB10 andpembrolizumab (anti-PD-1 antibody) were used in a mechanism-of-actionbased PD-1/TIGIT Combination Bioassay. Concentrations for each antibodywere 25, 10, 4, 1.6, 0.64, 0.256, 0.1024, 0.04096, and 0.016384 μg/ml.An untargeted IgG4 was used as a control. As shown in FIG. 9A, only thecombination of MAB10 and pembrolizumab (EC₅₀ of 5.06 nM) blocked bindingsufficiently to induce luciferase activity in the Jurkat cells. FIG. 9Bshows the results of the assay with a fixed dose (1 μg/ml) ofpembrolizumab (or IgG4 control) and a varying dose of MAB10 (50, 20, 8,3.2, 1.28, 0.512, 0.2048, 0.08192, and 0.032768 μg/ml). In FIGS. 9A and9B, neither the IgG4 control alone or the IgG4+MAB10 combination inducedluciferase activity.

FIGS. 10A-10D are a series of graphs showing the effect of MAB10 onCMV-stimulated CD4+ T-cells from a human donor using intracellularcytokine staining. Incubation of CD4+ cells with MAB10 (black bars)increases the production of the effector cytokines in a dose-dependentmatter, including TNF (FIG. 10A), IL-2 (FIG. 10B), and IFN-γ (FIG. 10C)compared to cells incubated with the IgG4 control (white bars). FIG. 10Dshows that incubation with MAB10 increases the proportion ofantigen-specific activated CD4+ T-cells. In FIG. 10D, cells that weretreated with 20 μg/ml of the IgG4 control or MAB10 were analyzed by FACSfor expression of CD3 (a marker of mature T-cells) and expression of TNFand IL-2. Statistical differences were calculated between MAB10 and IgG4control groups (same concentration treatments) using Student's T test(*=p<0.05, **=p<0.01, ***=p<0.005, ****=p<0.001).

FIGS. 11A-11D are a series of graphs similar to FIG. 10, but using CD8+cells, and show the production of TNF (FIG. 11A), perforin (FIG. 11B),and granzyme B (FIG. 11C) by such cells treated with MAB10 or the IgG4control. FIG. 11D shows that incubation with MAB10 also increases theproportion of antigen-specific activated CD8+ T-cells. Cells that weretreated with 20 μg/ml of the IgG4 control or MAB10 were analyzed by FACSfor expression of CD3 and expression of perforin and granzyme B.Statistical differences were calculated between MAB10 and IgG4 controlgroups (same concentration treatments) using Student's T test (*=p<0.05,**=p<0.01, ***=p<0.005, ****=p<0.001).

FIGS. 12A-12D show the results of treatment of cells from the samedonor, wherein blockade by MAB10 amplifies CMV-specific CD8+ T-cellresponses. Cells were incubated with a range of concentrations of MAB10(black bars) or the IgG4 control (white bars), and the percentage ofdouble positive population perforin+granzyme B+(FIG. 12A) or IFN-γ+TNF+(FIG. 12C) was analyzed. Statistical differences were calculated betweenMAB10 and IgG4 control groups (same concentration treatments) usingStudent's T test (*=p<0.05, **=p<0.01, ***=p<0.005, ****=p<0.001). FIG.12B (perforin+granzyme B+ analysis) and FIG. 12D (IFN-γ+TNF+ analysis)show the proportion of double positive cells comparing cells treatedwith 20 μg/ml of the control antibody (left panels) or 20 μg/ml of MAB10(right panels).

FIG. 13 is a graph showing the combinatorial effect of MAB10 and thePD-1 antibody pembrolizumab on cells from the same donor as used forFIGS. 10-12. Cells were stimulated with CMV lysates and treated with 2μg/ml pembrolizumab or control IgG4, and 10, 20, or 40 μg/ml controlantibody or MAB10, and production of TNF was measured. Four groups ofcells were tested, and treated with IgG4 control (white bars, left mostgroup), a constant amount of IgG4 control and a titration of MAB10 (darkgray bars, second group from left), a constant amount of pembrolizumaband a titration of IgG4 control (light gray bars, second group fromright), or a constant amount of pembrolizumab and a titration of MAB10(black bars, right hand group).

FIGS. 14A-14C are a series of graphs showing the effect ofMAB10+pembrolizumab treatment on cells from three different donors.Cells were stimulated with 0.1 μg/ml of CMV lysate and treated with 20μg/ml of MAB10 or 20 μg/ml of control IgG4 antibody and a titration ofpembrolizumab, then the production of TNF was measured. FIG. 14A showsthe results of the assay using cells from Donor 1; FIG. 14B shows theresults of the assay using cells from Donor 2; FIG. 14C shows theresults of the assay using cells from Donor 3. The addition of MAB10(black bars) alone or in combination with increasing concentrations ofpembrolizumab results in a greater production of TNF compared to controlantibody+pembrolizumab group (white bars). Additionally, MAB10 (blackbars) in combination with pembrolizumab also resulted in increasedactivation compared to MAB10 alone. Statistic differences werecalculated between MAB10 alone and MAB10+pembro groups using Student Ttest analysis (*=p<0.05, **=p<0.01, ***=p<0.005, ****=p<0.001)

DETAILED DESCRIPTION 1. Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a difference over what is generally understood in the art. Thetechniques and procedures described or referenced herein are generallywell understood and commonly employed using conventional methodologiesby those skilled in the art, such as, for example, the widely utilizedmolecular cloning methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 4th ed. (2012) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. As appropriate, proceduresinvolving the use of commercially available kits and reagents aregenerally carried out in accordance with manufacturer-defined protocolsand conditions unless otherwise noted.

As used herein, the singular forms “a,” “an,” and “the” include theplural referents unless the context clearly indicates otherwise. Theterms “include,” “such as,” and the like are intended to conveyinclusion without limitation, unless otherwise specifically indicated.

As used herein, the term “comprising” also specifically includesembodiments “consisting of” and “consisting essentially of” the recitedelements, unless specifically indicated otherwise. For example, amultispecific ABP “comprising a diabody” includes a multispecific ABP“consisting of a diabody” and a multispecific ABP “consistingessentially of a diabody.”

The term “about” indicates and encompasses an indicated value and arange above and below that value. In certain embodiments, the term“about” indicates the designated value ±10%, ±5%, or ±1%. In certainembodiments, where applicable, the term “about” indicates the designatedvalue(s)±one standard deviation of that value(s).

The terms “TIGIT,” “TIGIT protein,” and “TIGIT antigen” are usedinterchangeably herein to refer to human TIGIT, or any variants (e.g.,splice variants and allelic variants), isoforms, and species homologs ofhuman TIGIT that are naturally expressed by cells, or that are expressedby cells transfected with a tigit gene. In some aspects, the TIGITprotein is a TIGIT protein naturally expressed by a primate (e.g., amonkey or a human), a rodent (e.g., a mouse or a rat), a dog, a camel, acat, a cow, a goat, a horse, or a sheep. In some aspects, the TIGITprotein is human TIGIT (hTIGIT; SEQ ID NO:1). Without being bound bytheory, it is believed that positions 1-21 of SEQ ID NO:1 encode asignal peptide; positions 22-141 of SEQ ID NO:1 encode the extracellulardomain of the mature TIGIT protein; positions 142-162 of SEQ ID NO:1encode a transmembrane domain; and positions 163-244 of SEQ ID NO:1encode a cytoplasmic domain. See UniProt KB-Q495A1 (TIGIT_HUMAN), atwww.uniprot.org/uniprot/Q495A1, accessed Sep. 28, 2015. In some aspects,the TIGIT protein is a cynomolgus monkey TIGIT (cTIGIT; SEQ ID NO:2). Insome aspects, the TIGIT protein is a murine TIGIT (mTIGIT) having thesequence provided in SEQ ID NO:3. In some aspects, the TIGIT protein isa murine TIGIT (mTIGIT) having the sequence provided in SEQ ID NO:138.As used herein, if a SEQ ID NO is not specified, the terms “mTIGIT,”“murine TIGIT” and “mouse TIGIT mean SEQ ID NO: 3 and/or SEQ ID NO: 138.In some aspects, the TIGIT protein is a full-length or unprocessed TIGITprotein. In some aspects, the TIGIT protein is a truncated or processedTIGIT protein produced by post-translational modification. TIGIT is alsoknown by a variety of synonyms, including WUCAM, VSIG9, and Vstm3.

The term “immunoglobulin” refers to a class of structurally relatedproteins generally comprising two pairs of polypeptide chains: one pairof light (L) chains and one pair of heavy (H) chains. In an “intactimmunoglobulin,” all four of these chains are interconnected bydisulfide bonds. The structure of immunoglobulins has been wellcharacterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5(2013) Lippincott Williams & Wilkins, Philadelphia, Pa. Briefly, eachheavy chain typically comprises a heavy chain variable region (V_(H))and a heavy chain constant region (C_(H)). The heavy chain constantregion typically comprises three domains, abbreviated C_(H1), C_(H2),and C_(H3). Each light chain typically comprises a light chain variableregion (V_(L)) and a light chain constant region. The light chainconstant region typically comprises one domain, abbreviated C_(L).

The term “antigen-binding protein” (ABP) refers to a protein comprisingone or more antigen-binding domains that specifically bind to an antigenor epitope. In some embodiments, the antigen-binding domain binds theantigen or epitope with specificity and affinity similar to that ofnaturally occurring antibodies. In some embodiments, the ABP comprisesan antibody. In some embodiments, the ABP consists of an antibody. Insome embodiments, the ABP consists essentially of an antibody. In someembodiments, the ABP comprises an alternative scaffold. In someembodiments, the ABP consists of an alternative scaffold. In someembodiments, the ABP consists essentially of an alternative scaffold. Insome embodiments, the ABP comprises an antibody fragment. In someembodiments, the ABP consists of an antibody fragment. In someembodiments, the ABP consists essentially of an antibody fragment. A“TIGIT ABP,” “anti-TIGIT ABP,” or “TIGIT-specific ABP” is an ABP, asprovided herein, which specifically binds to the antigen TIGIT. In someembodiments, the ABP binds the extracellular domain of TIGIT. In certainembodiments, a TIGIT ABP provided herein binds to an epitope of TIGITthat is conserved between or among TIGIT proteins from differentspecies.

The term “antibody” is used herein in its broadest sense and includescertain types of immunoglobulin molecules comprising one or moreantigen-binding domains that specifically bind to an antigen or epitope.An antibody specifically includes intact antibodies (e.g., intactimmunoglobulins), antibody fragments, and multi-specific antibodies. Anantibody is one type of ABP.

The term “alternative scaffold” refers to a molecule in which one ormore regions may be diversified to produce one or more antigen-bindingdomains that specifically bind to an antigen or epitope. In someembodiments, the antigen-binding domain binds the antigen or epitopewith specificity and affinity similar to that of an antibody. Exemplaryalternative scaffolds include those derived from fibronectin (e.g.,Adnectins™), the β-sandwich (e.g., iMab), lipocalin (e.g., Anticalins®),EETI-II/AGRP, BPTI/LACI-D1/ITI-D2 (e.g., Kunitz domains), thioredoxinpeptide aptamers, protein A (e.g., Affibody®), ankyrin repeats (e.g.,DARPins), gamma-B-crystallin/ubiquitin (e.g., Affilins), CTLD₃ (e.g.,Tetranectins), Fynomers, and (LDLR-A module) (e.g., Avimers). Additionalinformation on alternative scaffolds is provided in Binz et al., Nat.Biotechnol., 2005 23:1257-1268; Skerra, Current Opin. in Biotech., 200718:295-304; and Silacci et al., J. Biol. Chem., 2014, 289:14392-14398;each of which is incorporated by reference in its entirety. Analternative scaffold is one type of ABP.

The term “antigen-binding domain” means the portion of an ABP that iscapable of specifically binding to an antigen or epitope. One example ofan antigen-binding domain is an antigen-binding domain formed by aV_(H)-V_(L) dimer of an antibody. Another example of an antigen-bindingdomain is an antigen-binding domain formed by diversification of certainloops from the tenth fibronectin type III domain of an Adnectin.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a naturally occurring antibodystructure and having heavy chains that comprise an Fc region. Forexample, when used to refer to an IgG molecule, a “full length antibody”is an antibody that comprises two heavy chains and two light chains.

The term “Fc region” means the C-terminal region of an immunoglobulinheavy chain that, in naturally occurring antibodies, interacts with Fcreceptors and certain proteins of the complement system. The structuresof the Fc regions of various immunoglobulins, and the glycosylationsites contained therein, are known in the art. See Schroeder andCavacini, J. Allergy Clin. Immunol., 2010, 125:S41-52, incorporated byreference in its entirety. The Fc region may be a naturally occurring Fcregion, or an Fc region modified as described in the art or elsewhere inthis disclosure.

The V_(H) and V_(L) regions may be further subdivided into regions ofhypervariability (“hypervariable regions (HVRs);” also called“complementarity determining regions” (CDRs)) interspersed with regionsthat are more conserved. The more conserved regions are called frameworkregions (FRs). Each V_(H) and V_(L) generally comprises three CDRs andfour FRs, arranged in the following order (from N-terminus toC-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved inantigen binding, and influence antigen specificity and binding affinityof the antibody. See Kabat et al., Sequences of Proteins ofImmunological Interest 5th ed. (1991) Public Health Service, NationalInstitutes of Health, Bethesda, Md., incorporated by reference in itsentirety.

The light chain from any vertebrate species can be assigned to one oftwo types, called kappa (κ) and lambda (λ), based on the sequence of itsconstant domain.

The heavy chain from any vertebrate species can be assigned to one offive different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. Theseclasses are also designated α, δ, ε, γ, and μ, respectively. The IgG andIgA classes are further divided into subclasses on the basis ofdifferences in sequence and function. Humans express the followingsubclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The amino acid sequence boundaries of a CDR can be determined by one ofskill in the art using any of a number of known numbering schemes,including those described by Kabat et al., supra (“Kabat” numberingscheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia”numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745(“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003,27:55-77 (“IMGT” numbering scheme); and Honegge and Plückthun, J. Mol.Biol., 2001, 309:657-70 (“AHo” numbering scheme); each of which isincorporated by reference in its entirety.

Table 1 provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1,CDR-H2, and CDR-H3 as identified by the Kabat and Chothia schemes. ForCDR-H1, residue numbering is provided using both the Kabat and Chothianumbering schemes.

CDRs may be assigned, for example, using antibody numbering software,such as Abnum, available at www.bioinf.org.uk/abs/abnum/, and describedin Abhinandan and Martin, Immunology, 2008, 45:3832-3839, incorporatedby reference in its entirety.

TABLE 1 Residues in CDRs according to Kabat and Chothia numberingschemes. CDR Kabat Chothia L1 L24-L34 L24-L34 L2 L50-L56 L50-L56 L3L89-L97 L89-L97 H1 (Kabat Numbering) H31-H35B H26-H32 or H34* H1(Chothia Numbering) H31-H35 H26-H32 H2 H50-H65 H52-H56 H3 H95-H102H95-H102 *The C-terminus of CDR-H1, when numbered using the Kabatnumbering convention, varies between H32 and H34, depending on thelength of the CDR.

The “EU numbering scheme” is generally used when referring to a residuein an antibody heavy chain constant region (e.g., as reported in Kabatet al., supra). Unless stated otherwise, the EU numbering scheme is usedto refer to residues in antibody heavy chain constant regions describedherein.

An “antibody fragment” comprises a portion of an intact antibody, suchas the antigen-binding or variable region of an intact antibody.Antibody fragments include, for example, Fv fragments, Fab fragments,F(ab′)₂ fragments, Fab′ fragments, scFv (sFv) fragments, and scFv-Fcfragments.

“Fv” fragments comprise a non-covalently-linked dimer of one heavy chainvariable domain and one light chain variable domain.

“Fab” fragments comprise, in addition to the heavy and light chainvariable domains, the constant domain of the light chain and the firstconstant domain (C_(H1)) of the heavy chain. Fab fragments may begenerated, for example, by recombinant methods or by papain digestion ofa full-length antibody.

“F(ab′)₂” fragments contain two Fab′ fragments joined, near the hingeregion, by disulfide bonds. F(ab′)₂ fragments may be generated, forexample, by recombinant methods or by pepsin digestion of an intactantibody. The F(ab′) fragments can be dissociated, for example, bytreatment with β-mercaptoethanol.

“Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise a V_(H)domain and a V_(L) domain in a single polypeptide chain. The V_(H) andV_(L) are generally linked by a peptide linker. See Plückthun A. (1994).Any suitable linker may be used. In some embodiments, the linker is a(GGGGS). (SEQ ID NO: 127). In some embodiments, n=1, 2, 3, 4, 5, or 6.See Antibodies from Escherichia coli. In Rosenberg M. & Moore G. P.(Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp.269-315). Springer-Verlag, New York, incorporated by reference in itsentirety.

“scFv-Fc” fragments comprise an scFv attached to an Fc domain. Forexample, an Fc domain may be attached to the C-terminal of the scFv. TheFc domain may follow the V_(H) or V_(L), depending on the orientation ofthe variable domains in the scFv (i.e., V_(H)-V_(L) or V_(L)-V_(H)). Anysuitable Fc domain known in the art or described herein may be used. Insome cases, the Fc domain comprises an IgG4 Fc domain.

The term “single domain antibody” refers to a molecule in which onevariable domain of an antibody specifically binds to an antigen withoutthe presence of the other variable domain. Single domain antibodies, andfragments thereof, are described in Arabi Ghahroudi et al., FEBSLetters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem.Sci., 2001, 26:230-245, each of which is incorporated by reference inits entirety. Single domain antibodies are also known as sdAbs ornanobodies.

A “multispecific ABP” is an ABP that comprises two or more differentantigen-binding domains that collectively specifically bind two or moredifferent epitopes. The two or more different epitopes may be epitopeson the same antigen (e.g., a single TIGIT molecule expressed by a cell)or on different antigens (e.g., different TIGIT molecules expressed bythe same cell, or a TIGIT molecule and a non-TIGIT molecule). In someaspects, a multi-specific ABP binds two different epitopes (i.e., a“bispecific ABP”). In some aspects, a multi-specific ABP binds threedifferent epitopes (i.e., a “trispecific ABP”). In some aspects, amulti-specific ABP binds four different epitopes (i.e., a “quadspecificABP”). In some aspects, a multi-specific ABP binds five differentepitopes (i.e., a “quintspecific ABP”). In some aspects, amulti-specific ABP binds 6, 7, 8, or more different epitopes. Eachbinding specificity may be present in any suitable valency. Examples ofmultispecific ABPs are provided elsewhere in this disclosure.

A “monospecific ABP” is an ABP that comprises one or more binding sitesthat specifically bind to a single epitope. An example of a monospecificABP is a naturally occurring IgG molecule which, while divalent (i.e.,having two antigen-binding domains), recognizes the same epitope at eachof the two antigen-binding domains. The binding specificity may bepresent in any suitable valency.

The term “monoclonal antibody” refers to an antibody from a populationof substantially homogeneous antibodies. A population of substantiallyhomogeneous antibodies comprises antibodies that are substantiallysimilar and that bind the same epitope(s), except for variants that maynormally arise during production of the monoclonal antibody. Suchvariants are generally present in only minor amounts. A monoclonalantibody is typically obtained by a process that includes the selectionof a single antibody from a plurality of antibodies. For example, theselection process can be the selection of a unique clone from aplurality of clones, such as a pool of hybridoma clones, phage clones,yeast clones, bacterial clones, or other recombinant DNA clones. Theselected antibody can be further altered, for example, to improveaffinity for the target (“affinity maturation”), to humanize theantibody, to improve its production in cell culture, and/or to reduceits immunogenicity in a subject.

The term “chimeric antibody” refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

“Humanized” forms of non-human antibodies are chimeric antibodies thatcontain minimal sequence derived from the non-human antibody. Ahumanized antibody is generally a human antibody (recipient antibody) inwhich residues from one or more CDRs are replaced by residues from oneor more CDRs of a non-human antibody (donor antibody). The donorantibody can be any suitable non-human antibody, such as a mouse, rat,rabbit, chicken, or non-human primate antibody having a desiredspecificity, affinity, or biological effect. In some instances, selectedframework region residues of the recipient antibody are replaced by thecorresponding framework region residues from the donor antibody.Humanized antibodies may also comprise residues that are not found ineither the recipient antibody or the donor antibody. Such modificationsmay be made to further refine antibody function. For further details,see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature,1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596,each of which is incorporated by reference in its entirety.

A “human antibody” is one which possesses an amino acid sequencecorresponding to that of an antibody produced by a human or a humancell, or derived from a non-human source that utilizes a human antibodyrepertoire or human antibody-encoding sequences (e.g., obtained fromhuman sources or designed de novo). Human antibodies specificallyexclude humanized antibodies.

An “isolated ABP” or “isolated nucleic acid” is an ABP or nucleic acidthat has been separated and/or recovered from a component of its naturalenvironment. Components of the natural environment may include enzymes,hormones, and other proteinaceous or nonproteinaceous materials. In someembodiments, an isolated ABP is purified to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence, for example by use of a spinning cup sequenator. In someembodiments, an isolated ABP is purified to homogeneity by gelelectrophoresis (e.g., SDS-PAGE) under reducing or nonreducingconditions, with detection by Coomassie blue or silver stain. In someembodiments, an isolated ABP may include an ABP in situ withinrecombinant cells, since at least one component of the ABP's naturalenvironment is not present. In some aspects, an isolated ABP or isolatednucleic acid is prepared by at least one purification step. In someembodiments, an isolated ABP or isolated nucleic acid is purified to atleast 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, anisolated ABP or isolated nucleic acid is purified to at least 80%, 85%,90%, 95%, or 99% by volume. In some embodiments, an isolated ABP orisolated nucleic acid is provided as a solution comprising at least 85%,90%, 95%, 98%, 99% to 100% ABP or nucleic acid by weight. In someembodiments, an isolated ABP or isolated nucleic acid is provided as asolution comprising at least 85%, 90%, 95%, 98%, 99% to 100% ABP ornucleic acid by volume.

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., an ABP)and its binding partner (e.g., an antigen or epitope). Unless indicatedotherwise, as used herein, “affinity” refers to intrinsic bindingaffinity, which reflects a 1:1 interaction between members of a bindingpair (e.g., ABP and antigen or epitope). The affinity of a molecule Xfor its partner Y can be represented by the dissociation equilibriumconstant (K_(D)). The kinetic components that contribute to thedissociation equilibrium constant are described in more detail below.Affinity can be measured by common methods known in the art, includingthose described herein, such as surface plasmon resonance (SPR)technology (e.g., BIACORE®) or biolayer interferometry (e.g.,FORTEBIO®).

With regard to the binding of an ABP to a target molecule, the terms“bind,” “specific binding,” “specifically binds to,” “specific for,”“selectively binds,” and “selective for” a particular antigen (e.g., apolypeptide target) or an epitope on a particular antigen mean bindingthat is measurably different from a non-specific or non-selectiveinteraction (e.g., with a non-target molecule). Specific binding can bemeasured, for example, by measuring binding to a target molecule andcomparing it to binding to a non-target molecule. Specific binding canalso be determined by competition with a control molecule that mimicsthe epitope recognized on the target molecule. In that case, specificbinding is indicated if the binding of the ABP to the target molecule iscompetitively inhibited by the control molecule. In some aspects, theaffinity of a TIGIT ABP for a non-target molecule is less than about 50%of the affinity for TIGIT. In some aspects, the affinity of a TIGIT ABPfor a non-target molecule is less than about 40% of the affinity forTIGIT. In some aspects, the affinity of a TIGIT ABP for a non-targetmolecule is less than about 30% of the affinity for TIGIT. In someaspects, the affinity of a TIGIT ABP for a non-target molecule is lessthan about 20% of the affinity for TIGIT. In some aspects, the affinityof a TIGIT ABP for a non-target molecule is less than about 10% of theaffinity for TIGIT. In some aspects, the affinity of a TIGIT ABP for anon-target molecule is less than about 1% of the affinity for TIGIT. Insome aspects, the affinity of a TIGIT ABP for a non-target molecule isless than about 0.1% of the affinity for TIGIT.

The term “k_(d)” (sec⁻¹), as used herein, refers to the dissociationrate constant of a particular ABP-antigen interaction. This value isalso referred to as the k_(off) value.

The term “k_(a)” (M⁻¹×sec⁻¹), as used herein, refers to the associationrate constant of a particular ABP-antigen interaction. This value isalso referred to as the k_(on) value.

The term “K_(D)” (M), as used herein, refers to the dissociationequilibrium constant of a particular ABP-antigen interaction.K_(D)=k_(d)/k_(a). In some embodiments, the affinity of an ABP isdescribed in terms of the K_(D) for an interaction between such ABP andits antigen. For clarity, as known in the art, a smaller K_(D) valueindicates a higher affinity interaction, while a larger K_(D) valueindicates a lower affinity interaction.

The term “K_(A)” (M⁻¹), as used herein, refers to the associationequilibrium constant of a particular ABP-antigen interaction.K_(A)=k_(a)/k_(d).

An “affinity matured” ABP is an ABP with one or more alterations (e.g.,in one or more CDRs or FRs) relative to a parent ABP (i.e., an ABP fromwhich the altered ABP is derived or designed) that result in animprovement in the affinity of the ABP for its antigen, compared to theparent ABP which does not possess the alteration(s). In someembodiments, an affinity matured ABP has nanomolar or picomolar affinityfor the target antigen. Affinity matured ABPs may be produced using avariety of methods known in the art. For example, Marks et al.(Bio/Technology, 1992, 10:779-783, incorporated by reference in itsentirety) describes affinity maturation by V_(H) and V_(L) domainshuffling. Random mutagenesis of CDR and/or framework residues isdescribed by, for example, Barbas et al. (Proc. Nat. Acad. Sci. U.S.A.,1994, 91:3809-3813); Schier et al., Gene, 1995, 169:147-155; Yelton etal., J. Immunol., 1995, 155:1994-2004; Jackson et al., J. Immunol.,1995, 154:3310-33199; and Hawkins et al, J. Mol. Biol., 1992,226:889-896; each of which is incorporated by reference in its entirety.

An “immunoconjugate” is an ABP conjugated to one or more heterologousmolecule(s), such as a therapeutic or diagnostic agent.

“Effector functions” refer to those biological activities mediated bythe Fc region of an antibody, which activities may vary depending on theantibody isotype. Examples of antibody effector functions include C1qbinding to activate complement dependent cytotoxicity (CDC), Fc receptorbinding to activate antibody-dependent cellular cytotoxicity (ADCC), andantibody dependent cellular phagocytosis (ADCP).

When used herein in the context of two or more ABPs, the term “competeswith” or “cross-competes with” indicates that the two or more ABPscompete for binding to an antigen (e.g., TIGIT). In one exemplary assay,TIGIT is coated on a surface and contacted with a first TIGIT ABP, afterwhich a second TIGIT ABP is added. In another exemplary assay, a firstTIGIT ABP is coated on a surface and contacted with TIGIT, and then asecond TIGIT ABP is added. If the presence of the first TIGIT ABPreduces binding of the second TIGIT ABP, in either assay, then the ABPscompete with each other. The term “competes with” also includescombinations of ABPs where one ABP reduces binding of another ABP, butwhere no competition is observed when the ABPs are added in the reverseorder. However, in some embodiments, the first and second ABPs inhibitbinding of each other, regardless of the order in which they are added.In some embodiments, one ABP reduces binding of another ABP to itsantigen by at least 25%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, or at least 95%. A skilledartisan can select the concentrations of the antibodies used in thecompetition assays based on the affinities of the ABPs for TIGIT and thevalency of the ABPs. The assays described in this definition areillustrative, and a skilled artisan can utilize any suitable assay todetermine if antibodies compete with each other. Suitable assays aredescribed, for example, in Cox et al., “Immunoassay Methods,” in AssayGuidance Manual [Internet], Updated Dec. 24, 2014(www.ncbi.nlm.nih.gov/books/NBK92434/; accessed Sep. 29, 2015); Silmanet al., Cytometry, 2001, 44:30-37; and Finco et al., J. Pharm. Biomed.Anal., 2011, 54:351-358; each of which is incorporated by reference inits entirety.

The term “epitope” means a portion of an antigen that specifically bindsto an ABP. Epitopes frequently consist of surface-accessible amino acidresidues and/or sugar side chains and may have specific threedimensional structural characteristics, as well as specific chargecharacteristics. Conformational and non-conformational epitopes aredistinguished in that the binding to the former but not the latter maybe lost in the presence of denaturing solvents. An epitope may compriseamino acid residues that are directly involved in the binding, and otheramino acid residues, which are not directly involved in the binding. Theepitope to which an ABP binds can be determined using known techniquesfor epitope determination such as, for example, testing for ABP bindingto TIGIT variants with different point-mutations, or to chimeric TIGITvariants.

Percent “identity” between a polypeptide sequence and a referencesequence, is defined as the percentage of amino acid residues in thepolypeptide sequence that are identical to the amino acid residues inthe reference sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent amino acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA,or MUSCLE software. Those skilled in the art can determine appropriateparameters for aligning sequences, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared.

A “conservative substitution” or a “conservative amino acidsubstitution,” refers to the substitution an amino acid with achemically or functionally similar amino acid. Conservative substitutiontables providing similar amino acids are well known in the art. By wayof example, the groups of amino acids provided in Tables 2-4 are, insome embodiments, considered conservative substitutions for one another.

TABLE 2 Selected groups of amino acids that are considered conservativesubstitutions for one another, in certain embodiments. Acidic Residues Dand E Basic Residues K, R, and H Hydrophilic Uncharged Residues S, T, N,and Q Aliphatic Uncharged Residues G, A, V, L, and I Non-polar UnchargedResidues C, M, and P Aromatic Residues F, Y, and W

TABLE 3 Additional selected groups of amino acids that are consideredconservative substitutions for one another, in certain embodiments.Group 1 A, S, and T Group 2 D and E Group 3 N and Q Group 4 R and KGroup 5 I, L, and M Group 6 F, Y, and W

TABLE 4 Further selected groups of amino acids that are consideredconservative substitutions for one another, in certain embodiments.Group A A and G Group B D and E Group C N and Q Group D R, K, and HGroup E I, L, M, V Group F F, Y, and W Group G S and T Group H C and M

Additional conservative substitutions may be found, for example, inCreighton, Proteins: Structures and Molecular Properties 2nd ed. (1993)W. H. Freeman & Co., New York, N.Y. An ABP generated by making one ormore conservative substitutions of amino acid residues in a parent ABPis referred to as a “conservatively modified variant.”

The term “amino acid” refers to the twenty common naturally occurringamino acids. Naturally occurring amino acids include alanine (Ala; A),arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), Glycine (Gly; G);histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys;K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P),serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr;Y), and valine (Val; V).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which an exogenous nucleicacid has been introduced, and the progeny of such cells. Host cellsinclude “transformants” (or “transformed cells”) and “transfectants” (or“transfected cells”), which each include the primary transformed ortransfected cell and progeny derived therefrom. Such progeny may not becompletely identical in nucleic acid content to a parent cell, and maycontain mutations.

The term “treating” (and variations thereof such as “treat” or“treatment”) refers to clinical intervention in an attempt to alter thenatural course of a disease or condition in a subject in need thereof.Treatment can be performed both for prophylaxis and during the course ofclinical pathology. Desirable effects of treatment include preventingoccurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to an amount of an ABP or pharmaceuticalcomposition provided herein that, when administered to a subject, iseffective to treat a disease or disorder.

As used herein, the term “subject” means a mammalian subject. Exemplarysubjects include humans, monkeys, dogs, cats, mice, rats, cows, horses,camels, goats, rabbits, and sheep. In certain embodiments, the subjectis a human. In some embodiments the subject has a disease or conditionthat can be treated with an ABP provided herein. In some aspects, thedisease or condition is a cancer. In some aspects, the disease orcondition is a viral infection.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic or diagnostic products(e.g., kits) that contain information about the indications, usage,dosage, administration, combination therapy, contraindications and/orwarnings concerning the use of such therapeutic or diagnostic products.

The term “cytotoxic agent,” as used herein, refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction.

A “chemotherapeutic agent” refers to a chemical compound useful in thetreatment of cancer. Chemotherapeutic agents include “anti-hormonalagents” or “endocrine therapeutics” which act to regulate, reduce,block, or inhibit the effects of hormones that can promote the growth ofcancer.

The term “cytostatic agent” refers to a compound or composition whicharrests growth of a cell either in vitro or in vivo. In someembodiments, a cytostatic agent is an agent that reduces the percentageof cells in S phase. In some embodiments, a cytostatic agent reduces thepercentage of cells in S phase by at least about 20%, at least about40%, at least about 60%, or at least about 80%.

The term “tumor” refers to all neoplastic cell growth and proliferation,whether malignant or benign, and all pre-cancerous and cancerous cellsand tissues. The terms “cancer,” “cancerous,” “cell proliferativedisorder,” “proliferative disorder” and “tumor” are not mutuallyexclusive as referred to herein. The terms “cell proliferative disorder”and “proliferative disorder” refer to disorders that are associated withsome degree of abnormal cell proliferation. In some embodiments, thecell proliferative disorder is a cancer. In some aspects, the tumor is asolid tumor. In some aspects, the tumor is a hematologic malignancy.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective in treating a subject, andwhich contains no additional components which are unacceptably toxic tothe subject in the amounts provided in the pharmaceutical composition.

The terms “modulate” and “modulation” refer to reducing or inhibitingor, alternatively, activating or increasing, a recited variable.

The terms “increase” and “activate” refer to an increase of 10%, 20%,30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold,4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in arecited variable.

The terms “reduce” and “inhibit” refer to a decrease of 10%, 20%, 30%,40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recitedvariable.

The term “agonize” refers to the activation of receptor signaling toinduce a biological response associated with activation of the receptor.An “agonist” is an entity that binds to and agonizes a receptor.

The term “antagonize” refers to the inhibition of receptor signaling toinhibit a biological response associated with activation of thereceptor. An “antagonist” is an entity that binds to and antagonizes areceptor.

The term “effector T cell” includes T helper (i.e., CD4+) cells andcytotoxic (i.e., CD8+) T cells. CD4+ effector T cells contribute to thedevelopment of several immunologic processes, including maturation of Bcells into plasma cells and memory B cells, and activation of cytotoxicT cells and macrophages. CD8+ effector T cells destroy virus-infectedcells and tumor cells. See Seder and Ahmed, Nature Immunol., 2003,4:835-842, incorporated by reference in its entirety, for additionalinformation on effector T cells.

The term “regulatory T cell” includes cells that regulate immunologicaltolerance, for example, by suppressing effector T cells. In someaspects, the regulatory T cell has a CD4+CD25+Foxp3+ phenotype. In someaspects, the regulatory T cell has a CD8+CD25+ phenotype. See Nocentiniet al., Br. J. Pharmacol., 2012, 165:2089-2099, incorporated byreference in its entirety, for additional information on regulatory Tcells expressing TIGIT.

The term “dendritic cell” refers to a professional antigen-presentingcell capable of activating a naïve T cell and stimulating growth anddifferentiation of a B cell.

2. TIGIT Antigen-Binding Proteins

2.1. TIGIT Binding and Target Cells

Provided herein are ABPs that specifically bind to TIGIT. In someaspects, the TIGIT is hTIGIT (SEQ ID NO:1). In some aspects, the TIGITis cTIGIT (SEQ ID NO:2). In some aspects, the TIGIT is mTIGIT with thesequence provided in SEQ ID NO:3. In some aspects, the TIGIT is mTIGITwith the sequence provided in SEQ ID NO:138.

In some embodiments, the ABPs provided herein specifically bind tohTIGIT (SEQ ID NO:1), cTIGIT (SEQ ID NO:2), and mTIGIT of SEQ ID NO:3.In some embodiments, the ABPs provided herein specifically bind tohTIGIT (SEQ ID NO:1), cTIGIT (SEQ ID NO:2), and mTIGIT of SEQ ID NO:138.In some embodiments, the ABPs provided herein specifically bind tohTIGIT (SEQ ID NO:1), and cTIGIT (SEQ ID NO:2). In some embodiments, theABPs provided herein specifically bind to hTIGIT (SEQ ID NO:1). In someembodiments, the ABPs provided herein do not bind mTIGIT of SEQ ID NO:3.In some embodiments, the ABPs provided herein do not bind mTIGIT of SEQID NO:138.

In some embodiments, the ABPs provided herein specifically bind to theextracellular domain of TIGIT.

In some embodiments, an ABP provided herein is an antibody. In someembodiments, an ABP provided herein is an antibody fragment. In someembodiments, an ABP provided herein is an alternative scaffold.

The TIGIT may be expressed on the surface of any suitable target cell.In some embodiments, the target cell is a T cell. In some embodiments,the target cell is an effector T cell. In some embodiments, the targetcell is a regulatory T cell. In some embodiments, the target cell is anatural killer (NK) cell. In some embodiments, the target cell is anatural killer T (NKT) cell.

In some embodiments, the ABPs provided herein comprise an immunoglobulinmolecule. In some embodiments, the ABPs provided herein consist of animmunoglobulin molecule. In some embodiments, the ABPs provided hereinconsist essentially of an immunoglobulin molecule. In some aspects, theimmunoglobulin molecule comprises an antibody. In some aspects, theimmunoglobulin molecule consists of an antibody. In some aspects, theimmunoglobulin molecule consists essentially of an antibody.

In some embodiments, the ABPs provided herein comprise a light chain. Insome aspects, the light chain is a kappa light chain. In some aspects,the light chain is a lambda light chain.

In some embodiments, the ABPs provided herein comprise a kappa lightchain comprising SEQ ID NO: 126.

In some embodiments, the ABPs provided herein comprise a heavy chain. Insome aspects, the heavy chain is an IgA. In some aspects, the heavychain is an IgD. In some aspects, the heavy chain is an IgE. In someaspects, the heavy chain is an IgG. In some aspects, the heavy chain isan IgM. In some aspects, the heavy chain is an IgG1. In some aspects,the heavy chain is an IgG2. In some aspects, the heavy chain is an IgG3.In some aspects, the heavy chain is an IgG4. In some aspects, the heavychain is an IgA1. In some aspects, the heavy chain is an IgA2.

In some embodiments, the ABPs provided herein comprise an IgG4 heavychain comprising a sequence selected from SEQ ID NO:55 and SEQ ID NO:56.In some embodiments, the ABPs provided herein comprise an IgG1 heavychain comprising a sequence selected from SEQ ID NO:57 and SEQ ID NO:125.

In some embodiments, the ABPs provided herein comprise an antibodyfragment. In some embodiments, the ABPs provided herein consist of anantibody fragment. In some embodiments, the ABPs provided herein consistessentially of an antibody fragment. In some aspects, the antibodyfragment is an Fv fragment. In some aspects, the antibody fragment is aFab fragment. In some aspects, the antibody fragment is a F(ab′)₂fragment. In some aspects, the antibody fragment is a Fab′ fragment. Insome aspects, the antibody fragment is an scFv (sFv) fragment. In someaspects, the antibody fragment is an scFv-Fc fragment. In some aspects,the antibody fragment is a fragment of a single domain antibody.

In some embodiments, an antibody fragment provided herein is derivedfrom an illustrative antibody provided herein. In some embodiments, anantibody fragments provided herein is not derived from an illustrativeantibody provided herein and may, for example, be isolated de novoaccording to the methods provided herein for obtaining antibodyfragments.

In some embodiments, an antibody fragment provided specifically bindshTIGIT. In some embodiments, an antibody fragment provided hereinspecifically binds cTIGIT. In some embodiments, an antibody fragmentprovided herein specifically binds mTIGIT. In some embodiments, anantibody fragment provided herein specifically binds hTIGIT and cTIGIT.In some embodiments, an antibody fragment provided herein specificallybinds hTIGIT and mTIGIT. In some embodiments, an antibody fragmentprovided herein specifically binds cTIGIT and mTIGIT. In someembodiments, an antibody fragment provided herein specifically bindshTIGIT, cTIGIT and mTIGIT.

In some embodiments, an antibody fragment derived from an antibodyprovided herein retains affinity, as measured by K_(D), for hTIGIT thatis within about 1.5-fold, about 2-fold, about 3-fold, about 4-fold,about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold orabout 10-fold the affinity of such antibody. In some embodiments, anantibody fragment derived from an antibody provided herein retainsaffinity, as measured by K_(D), for cTIGIT that is within about1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about6-fold, about 7-fold, about 8-fold, about 9-fold or about 10-fold theaffinity of such antibody. In some embodiments, an antibody fragmentderived from an antibody provided herein retains affinity, as measuredby K_(D), for mTIGIT that is within about 1.5-fold, about 2-fold, about3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about8-fold, about 9-fold or about 10-fold the affinity of such antibody. Insome embodiments, an antibody fragment derived from an antibody providedherein retains affinity, as measured by K_(D), for both hTIGIT andcTIGIT that is within about 1.5-fold, about 2-fold, about 3-fold, about4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about9-fold or about 10-fold the affinity of such antibody. In someembodiments, an antibody fragment derived from an antibody providedherein retains affinity, as measured by K_(D), for both hTIGIT andmTIGIT that is within about 1.5-fold, about 2-fold, about 3-fold, about4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about9-fold or about 10-fold the affinity of such antibody. In someembodiments, an antibody fragment derived from an antibody providedherein retains affinity, as measured by K_(D), for both cTIGIT andmTIGIT that is within about 1.5-fold, about 2-fold, about 3-fold, about4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about9-fold or about 10-fold the affinity of such antibody. In someembodiments, an antibody fragment derived from an antibody providedherein retains affinity, as measured by K_(D), for all three of hTIGIT,cTIGIT and mTIGIT that is within about 1.5-fold, about 2-fold, about3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about8-fold, about 9-fold or about 10-fold the affinity of such antibody.

In some embodiments, an antibody fragment provided herein retains theability to antagonize TIGIT, as measured by one or more assays orbiological effects described herein. In some embodiments, an antibodyfragment provided herein retains the ability to prevent TIGIT frominteracting with one or more of its ligands, as described herein.

In some embodiments, an antibody fragment provided herein competes forbinding to TIGIT with an antibody selected from MAB1, MAB2, MAB3, MAB4,MAB5, MAB6, MAB7, MAB8, MAB9, MAB10, MAB11, MAB12, MAB13, MAB14, MAB15,MAB16, MAB17, MAB18, MAB19, MAB20, or MAB21, each as provided in Table 5of this disclosure.

In some embodiments, an antibody fragment provided herein inhibitsbinding of CD155 to TIGIT. In some embodiments, an antibody fragmentprovided herein inhibits binding of CD112 to TIGIT. In some embodiments,an antibody fragment provided herein inhibits association of CD226 withTIGIT.

In some embodiments, an antibody fragment provided herein activates aneffector T cell or a natural killer (NK) cell. In some embodiments, anantibody fragment provided herein decreases the number of regulatory Tcells in a tissue or in circulation. In some embodiments, an antibodyfragment provided herein inhibits the suppression of an effector T cellby a regulatory T cell.

In some embodiments, an antibody fragment provided herein does not bindspecifically to any of PVRL1, PVRL2, PVRL3, or PVRL4.

In some embodiments, an antibody fragment provided herein binds murineTIGIT (mTIGIT; SEQ ID NO: 3) with an affinity lower (as indicated byhigher K_(D)) than the affinity of the antibody fragment for hTIGIT, ordoes not bind mTIGIT.

In some embodiments, a fragment of an antibody provided herein binds thesame epitope of TIGIT as such antibody.

In some embodiments, the ABPs provided herein are monoclonal antibodies.In some embodiments, the ABPs provided herein are polyclonal antibodies.

In some embodiments, the ABPs provided herein comprise a chimericantibody. In some embodiments, the ABPs provided herein consist of achimeric antibody. In some embodiments, the ABPs provided herein consistessentially of a chimeric antibody. In some embodiments, the ABPsprovided herein comprise a humanized antibody. In some embodiments, theABPs provided herein consist of a humanized antibody. In someembodiments, the ABPs provided herein consist essentially of a humanizedantibody. In some embodiments, the ABPs provided herein comprise a humanantibody. In some embodiments, the ABPs provided herein consist of ahuman antibody. In some embodiments, the ABPs provided herein consistessentially of a human antibody.

In some embodiments, the ABPs provided herein are affinity matured. Insome aspects, the affinity matured ABPs are affinity matured ABPsderived from an illustrative ABP provided herein.

In some embodiments, the ABPs provided herein comprise an alternativescaffold. In some embodiments, the ABPs provided herein consist of analternative scaffold. In some embodiments, the ABPs provided hereinconsist essentially of an alternative scaffold. Any suitable alternativescaffold may be used. In some aspects, the alternative scaffold isselected from an Adnectin™, an iMab, an Anticalin®, an EETI-II/AGRP, aKunitz domain, a thioredoxin peptide aptamer, an Affibody®, a DARPin, anAffilin, a Tetranectin, a Fynomer, and an Avimer.

In some embodiments, an ABP provided herein inhibits binding of TIGIT toone or more ligands of TIGIT. In some aspects, the ligand of TIGIT isselected from one or more of poliovirus receptor (PVR; CD155) andnectin-2 (CD112, PVRL2). In some aspects, the ABP inhibits binding ofTIGIT to one or more ligands of TIGIT by at least about 50%. In someaspects, the ABP inhibits binding of TIGIT to one or more ligands ofTIGIT by at least about 75%. In some aspects, the ABP inhibits bindingof TIGIT to one or more ligands of TIGIT by at least about 90%. In someaspects, the ABP inhibits binding of TIGIT to one or more ligands ofTIGIT by at least about 95%.

In some embodiments, an ABP of the invention is an ABP that competeswith an illustrative ABP provided herein. In some aspects, the ABP thatcompetes with the illustrative ABP provided herein binds the sameepitope as an illustrative ABP provided herein.

In some embodiments, an ABP provided herein does not bind PVRL4.

It is known that when an antibody is expressed in cells, the antibody ismodified after translation. Examples of the posttranslationalmodification include cleavage of lysine at the C terminal of the heavychain by a carboxypeptidase; modification of glutamine or glutamic acidat the N terminal of the heavy chain and the light chain to pyroglutamicacid by pyroglutamylation; glycosylation; oxidation; deamidation; andglycation, and it is known that such posttranslational modificationsoccur in various antibodies (See Journal of Pharmaceutical Sciences,2008, Vol. 97, p. 2426-2447, incorporated by reference in its entirety).In some embodiments, an ABP of the invention is an antibody orantigen-binding fragment thereof which has undergone posttranslationalmodification. Examples of an antibody or antigen-binding fragmentthereof which have undergone posttranslational modification include anantibody or antigen-binding fragments thereof which have undergonepyroglutamylation at the N terminal of the heavy chain variable regionand/or deletion of lysine at the C terminal of the heavy chain. It isknown in the art that such posttranslational modification due topyroglutamylation at the N terminal and deletion of lysine at the Cterminal does not have any influence on the activity of the antibody orfragment thereof (Analytical Biochemistry, 2006, Vol. 348, p. 24-39,incorporated by reference in its entirety).

2.2. Sequences of TIGIT Antigen-Binding Proteins

2.2.1. V_(H) Domains

In some embodiments, an ABP provided herein comprises a V_(H) sequenceselected from SEQ ID NOs: 4-24. In some embodiments an ABP providedherein comprises a V_(H) sequence of SEQ ID NO:4. In some embodiments anABP provided herein comprises a V_(H) sequence of SEQ ID NO:5. In someembodiments an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:6. In some embodiments an ABP provided herein comprises a V_(H)sequence of SEQ ID NO:7. In some embodiments an ABP provided hereincomprises a V_(H) sequence of SEQ ID NO:8. In some embodiments an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:9. In someembodiments an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:10. In some embodiments an ABP provided herein comprises a V_(H)sequence of SEQ ID NO:11. In some embodiments an ABP provided hereincomprises a V_(H) sequence of SEQ ID NO:12. In some embodiments an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:13. In someembodiments an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:14. In some embodiments an ABP provided herein comprises a V_(H)sequence of SEQ ID NO:15. In some embodiments an ABP provided hereincomprises a V_(H) sequence of SEQ ID NO:16. In some embodiments an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:17. In someembodiments an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:18. In some embodiments an ABP provided herein comprises a V_(H)sequence of SEQ ID NO:19. In some embodiments an ABP provided hereincomprises a V_(H) sequence of SEQ ID NO:20. In some embodiments an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:21. In someembodiments an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:22. In some embodiments an ABP provided herein comprises a V_(H)sequence of SEQ ID NO:23. In some embodiments an ABP provided hereincomprises a V_(H) sequence of SEQ ID NO:24.

In some embodiments, an ABP provided herein comprises a V_(H) sequencehaving at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity toan illustrative V_(H) sequence provided in SEQ ID NOs: 4-24. In someembodiments, an ABP provided herein comprises a V_(H) sequence providedin SEQ ID NOs: 4-24, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acidsubstitutions. In some aspects, the amino acid substitutions areconservative amino acid substitutions. In some embodiments, the ABPsdescribed in this paragraph are referred to herein as “variants.” Insome embodiments, such variants are derived from a sequence providedherein, for example, by affinity maturation, site directed mutagenesis,random mutagenesis, or any other method known in the art or describedherein. In some embodiments, such variants are not derived from asequence provided herein and may, for example, be isolated de novoaccording to the methods provided herein for obtaining ABPs.

2.2.2. V_(L) Domains

In some embodiments, an ABP provided herein comprises a V_(L) sequenceselected from SEQ ID NOs: 25-28. In some embodiments an ABP providedherein comprises a V_(L) sequence of SEQ ID NO:25. In some embodimentsan ABP provided herein comprises a V_(L) sequence of SEQ ID NO:26. Insome embodiments an ABP provided herein comprises a V_(L) sequence ofSEQ ID NO:27. In some embodiments an ABP provided herein comprises aV_(L) sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(L) sequencehaving at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity toan illustrative V_(L) sequence provided in SEQ ID NOs: 25-28. In someembodiments, an ABP provided herein comprises a V_(L) sequence providedin SEQ ID NOs: 25-28, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acidsubstitutions. In some aspects, the amino acid substitutions areconservative amino acid substitutions. In some embodiments, the ABPsdescribed in this paragraph are referred to herein as “variants.” Insome embodiments, such variants are derived from a sequence providedherein, for example, by affinity maturation, site directed mutagenesis,random mutagenesis, or any other method known in the art or describedherein. In some embodiments, such variants are not derived from asequence provided herein and may, for example, be isolated de novoaccording to the methods provided herein for obtaining ABPs.

2.2.3. V_(H)-V_(L) Combinations

In some embodiments, an ABP provided herein comprises a V_(H) sequenceselected from SEQ ID NOs: 4-24 and a V_(L) sequence selected from SEQ IDNOs: 25-28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:4 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:5 and a V_(L) sequence of SEQ ID NO:25. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:6 and a V_(L)sequence of SEQ ID NO:25. In some embodiments, an ABP provided hereincomprises a V_(H) sequence of SEQ ID NO:7 and a V_(L) sequence of SEQ IDNO:25. In some embodiments, an ABP provided herein comprises a V_(H)sequence of SEQ ID NO:8 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:9 and a V_(L) sequence of SEQ ID NO:26. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:10 and a V_(L)sequence of SEQ ID NO:26. In some embodiments, an ABP provided hereincomprises a V_(H) sequence of SEQ ID NO:11 and a V_(L) sequence of SEQID NO:26. In some embodiments, an ABP provided herein comprises a V_(H)sequence of SEQ ID NO:12 and a V_(L) sequence of SEQ ID NO:26. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:13 and a V_(L) sequence of SEQ ID NO:26. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:14 and a V_(L)sequence of SEQ ID NO:26. In some embodiments, an ABP provided hereincomprises a V_(H) sequence of SEQ ID NO:15 and a V_(L) sequence of SEQID NO:26. In some embodiments, an ABP provided herein comprises a V_(H)sequence of SEQ ID NO:16 and a V_(L) sequence of SEQ ID NO:27. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:17 and a V_(L) sequence of SEQ ID NO:27. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:18 and a V_(L)sequence of SEQ ID NO:27. In some embodiments, an ABP provided hereincomprises a V_(H) sequence of SEQ ID NO:19 and a V_(L) sequence of SEQID NO:27. In some embodiments, an ABP provided herein comprises a V_(H)sequence of SEQ ID NO:20 and a V_(L) sequence of SEQ ID NO:27. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:21 and a V_(L) sequence of SEQ ID NO:27. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:22 and a V_(L)sequence of SEQ ID NO:28. In some embodiments, an ABP provided hereincomprises a V_(H) sequence of SEQ ID NO:23 and a V_(L) sequence of SEQID NO:28. In some embodiments, an ABP provided herein comprises a V_(H)sequence of SEQ ID NO:24 and a V_(L) sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:4 and a V_(L) sequence of SEQ ID NO:26. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:4 and a V_(L) sequence of SEQ ID NO:27. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:4 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:5 and a V_(L) sequence of SEQ ID NO:26. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:5 and a V_(L) sequence of SEQ ID NO:27. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:5 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:6 and a V_(L) sequence of SEQ ID NO:26. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:6 and a V_(L) sequence of SEQ ID NO:27. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:6 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:7 and a V_(L) sequence of SEQ ID NO:26. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:7 and a V_(L) sequence of SEQ ID NO:27. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:7 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:8 and a V_(L) sequence of SEQ ID NO:26. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:8 and a V_(L) sequence of SEQ ID NO:27. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:8 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:9 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:9 and a V_(L) sequence of SEQ ID NO:27. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:9 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:10 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:10 and a V_(L) sequence of SEQ ID NO:27. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:10 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:11 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:11 and a V_(L) sequence of SEQ ID NO:27. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:11 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:12 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:12 and a V_(L) sequence of SEQ ID NO:27. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:12 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:13 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:13 and a V_(L) sequence of SEQ ID NO:27. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:13 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:14 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:14 and a V_(L) sequence of SEQ ID NO:27. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:14 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:15 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:15 and a V_(L) sequence of SEQ ID NO:27. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:15 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:16 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:16 and a V_(L) sequence of SEQ ID NO:26. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:16 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:17 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:17 and a V_(L) sequence of SEQ ID NO:26. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:17 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:18 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:18 and a V_(L) sequence of SEQ ID NO:26. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:18 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:19 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:19 and a V_(L) sequence of SEQ ID NO:26. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:19 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:20 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:20 and a V_(L) sequence of SEQ ID NO:26. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:20 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:21 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:21 and a V_(L) sequence of SEQ ID NO:26. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:21 and a V_(L)sequence of SEQ ID NO:28.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:22 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:22 and a V_(L) sequence of SEQ ID NO:26. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:22 and a V_(L)sequence of SEQ ID NO:27.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:23 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:23 and a V_(L) sequence of SEQ ID NO:26. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:23 and a V_(L)sequence of SEQ ID NO:27.

In some embodiments, an ABP provided herein comprises a V_(H) sequenceof SEQ ID NO:24 and a V_(L) sequence of SEQ ID NO:25. In someembodiments, an ABP provided herein comprises a V_(H) sequence of SEQ IDNO:24 and a V_(L) sequence of SEQ ID NO:26. In some embodiments, an ABPprovided herein comprises a V_(H) sequence of SEQ ID NO:24 and a V_(L)sequence of SEQ ID NO:27.

In some embodiments, an ABP provided herein comprises a V_(H) sequencehaving at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity toan illustrative V_(H) sequence provided in SEQ ID NOs: 4-24, and a V_(L)sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99%identity to an illustrative V_(L) sequence provided in SEQ ID NOs:25-28. In some embodiments, an ABP provided herein comprises a V_(H)sequence provided in SEQ ID NOs: 4-24, with up to 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25amino acid substitutions, and a V_(L) sequence provided in SEQ ID NOs:25-28, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. In someaspects, the amino acid substitutions are conservative amino acidsubstitutions. In some embodiments, the ABPs described in this paragraphare referred to herein as “variants.” In some embodiments, such variantsare derived from a sequence provided herein, for example, by affinitymaturation, site directed mutagenesis, random mutagenesis, or any othermethod known in the art or described herein. In some embodiments, suchvariants are not derived from a sequence provided herein and may, forexample, be isolated de novo according to the methods provided hereinfor obtaining ABPs.

2.2.4. CDRs

In some embodiments, an ABP provided herein comprises one to three CDRsof a V_(H) domain selected from SEQ ID NOs: 4-24. In some embodiments,an ABP provided herein comprises two to three CDRs of a V_(H) domainselected from SEQ ID NOs: 4-24. In some embodiments, an ABP providedherein comprises three CDRs of a V_(H) domain selected from SEQ ID NOs:4-24. In some aspects, the CDRs are Kabat CDRs. In some aspects, theCDRs are Chothia CDRs. In some aspects, the CDRs are IMGT CDRs.

In some embodiments, the CDRs are CDRs having at least about 50%, 75%,80%, 85%, 90%, or 95% identity with a CDR-H1, CDR-H2, or CDR-H3 of SEQID NOs: 4-24. In some embodiments, the CDR-H1 is a CDR-H1 of a V_(H)domain selected from SEQ ID NOs: 4-24, with up to 1, 2, 3, 4, or 5 aminoacid substitutions. In some embodiments, the CDR-H2 is a CDR-H2 of aV_(H) domain selected from SEQ ID NOs: 4-24, with up to 1, 2, 3, 4, 5,6, 7, or 8 amino acid substitutions. In some embodiments, the CDR-H3 isa CDR-H3 of a V_(H) domain selected from SEQ ID NOs: 4-24, with up to 1,2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, theamino acid substitutions are conservative amino acid substitutions. Insome embodiments, the ABPs described in this paragraph are referred toherein as “variants.” In some embodiments, such variants are derivedfrom a sequence provided herein, for example, by affinity maturation,site directed mutagenesis, random mutagenesis, or any other method knownin the art or described herein. In some embodiments, such variants arenot derived from a sequence provided herein and may, for example, beisolated de novo according to the methods provided herein for obtainingABPs.

In some embodiments, an ABP provided herein comprises one to three CDRsof a V_(L) domain selected from SEQ ID NOs: 25-28. In some embodiments,an ABP provided herein comprises two to three CDRs of a V_(L) domainselected from SEQ ID NOs: 25-28. In some embodiments, an ABP providedherein comprises three CDRs of a V_(L) domain selected from SEQ ID NOs:25-28. In some aspects, the CDRs are Kabat CDRs. In some aspects, theCDRs are Chothia CDRs. In some aspects, the CDRs are IMGT CDRs.

In some embodiments, the CDRs are CDRs having at least about 50%, 75%,80%, 85%, 90%, or 95% identity with a CDR-L1, CDR-L2, or CDR-L3 of SEQID NOs: 25-28. In some embodiments, the CDR-L1 is a CDR-L1 of a V_(L)domain selected from SEQ ID NOs: 25-28, with up to 1, 2, 3, 4, 5, or 6amino acid substitutions. In some embodiments, the CDR-L2 is a CDR-L2 ofa V_(L) domain selected from SEQ ID NOs: 25-28, with up to 1, 2, 3, or 4amino acid substitutions. In some embodiments, the CDR-L3 is a CDR-L3 ofa V_(L) domain selected from SEQ ID NOs: 25-28, with up to 1, 2, 3, 4,or 5 amino acid substitutions. In some aspects, the amino acidsubstitutions are conservative amino acid substitutions. In someembodiments, the ABPs described in this paragraph are referred to hereinas “variants.” In some embodiments, such variants are derived from asequence provided herein, for example, by affinity maturation, sitedirected mutagenesis, random mutagenesis, or any other method known inthe art or described herein. In some embodiments, such variants are notderived from a sequence provided herein and may, for example, beisolated de novo according to the methods provided herein for obtainingABPs.

In some embodiments, an ABP provided herein comprises one to three CDRsof a V_(H) domain selected from SEQ ID NOs: 4-24 and one to three CDRsof a V_(L) domain selected from SEQ ID NOs: 25-28. In some embodiments,an ABP provided herein comprises two to three CDRs of a V_(H) domainselected from SEQ ID NOs: 4-24 and two to three CDRs of a V_(L) domainselected from SEQ ID NOs: 25-28. In some embodiments, an ABP providedherein comprises three CDRs of a V_(H) domain selected from SEQ ID NOs:4-24 and three CDRs of a V_(L) domain selected from SEQ ID NOs: 25-28.In some aspects, the CDRs are Kabat CDRs. In some aspects, the CDRs areChothia CDRs. In some aspects, the CDRs are IMGT CDRs.

In some embodiments, the CDRs are CDRs having at least about 50%, 75%,80%, 85%, 90%, or 95% identity with a CDR-H1, CDR-H2, or CDR-H3 of SEQID NOs: 4-24 and at least about 50%, 75%, 80%, 85%, 90%, or 95% identitywith a CDR-L1, CDR-L2, or CDR-L3 of SEQ ID NOs: 25-28. In someembodiments, the CDR-H1 is a CDR-H1 of a V_(H) domain selected from SEQID NOs: 4-24, with up to 1, 2, 3, 4, or 5 amino acid substitutions; theCDR-H2 is a CDR-H2 of a V_(H) domain selected from SEQ ID NOs: 4-24,with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; theCDR-H3 is a CDR-H3 of a V_(H) domain selected from SEQ ID NOs: 4-24,with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; theCDR-L1 is a CDR-L1 of a V_(L) domain selected from SEQ ID NOs: 25-28,with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions; the CDR-L2 is aCDR-L2 of a V_(L) domain selected from SEQ ID NOs: 25-28, with up to 1,2, 3, or 4 amino acid substitutions; and the CDR-L3 is a CDR-L3 of aV_(L) domain selected from SEQ ID NOs: 25-28, with up to 1, 2, 3, 4, or5 amino acid substitutions. In some aspects, the amino acidsubstitutions are conservative amino acid substitutions. In someembodiments, the ABPs described in this paragraph are referred to hereinas “variants.” In some embodiments, such variants are derived from asequence provided herein, for example, by affinity maturation, sitedirected mutagenesis, random mutagenesis, or any other method known inthe art or described herein. In some embodiments, such variants are notderived from a sequence provided herein and may, for example, beisolated de novo according to the methods provided herein for obtainingABPs.

In some embodiments, an ABP provided herein comprises a CDR-H3 selectedfrom SEQ ID NOs: 29-35. In some aspects, the CDR-H3 has at least about50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H3 of SEQ ID NOs:29-35. In some aspects, the CDR-H3 is a CDR-H3 according to the IMGTnumbering system. In some embodiments, the CDR-H3 is a CDR-H3 selectedfrom SEQ ID NOs: 29-35, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acidsubstitutions. In some aspects, the amino acid substitutions areconservative amino acid substitutions. In some embodiments, the ABPsdescribed in this paragraph are referred to herein as “variants.” Insome embodiments, such variants are derived from a sequence providedherein, for example, by affinity maturation, site directed mutagenesis,random mutagenesis, or any other method known in the art or describedherein. In some embodiments, such variants are not derived from asequence provided herein and may, for example, be isolated de novoaccording to the methods provided herein for obtaining ABPs.

In some embodiments, an ABP provided herein comprises a CDR-H2 selectedfrom SEQ ID NOs: 36-47. In some aspects, the CDR-H2 has at least about50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H2 of SEQ ID NOs:36-47. In some aspects, the CDR-H2 is a CDR-H2 according to the Kabatnumbering system. In some embodiments, the CDR-H2 is a CDR-H2 selectedfrom SEQ ID NOs: 36-47, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acidsubstitutions. In some aspects, the amino acid substitutions areconservative amino acid substitutions. In some embodiments, the ABPsdescribed in this paragraph are referred to herein as “variants.” Insome embodiments, such variants are derived from a sequence providedherein, for example, by affinity maturation, site directed mutagenesis,random mutagenesis, or any other method known in the art or describedherein. In some embodiments, such variants are not derived from asequence provided herein and may, for example, be isolated de novoaccording to the methods provided herein for obtaining ABPs.

In some embodiments, an ABP provided herein comprises a CDR-H1 selectedfrom SEQ ID NOs: 48-54 or 58-62. In some aspects, the CDR-H1 has atleast about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H1 ofSEQ ID NOs: 48-54 or 58-62. In some aspects, the CDR-H1 is a CDR-H1 thatspans the CDR-H1 as defined by both the Chothia and Kabat numberingsystems. In some embodiments, the CDR-H1 is a CDR-H1 selected from SEQID NOs: 48-54 or 58-62, with up to 1, 2, 3, 4, or 5 amino acidsubstitutions. In some aspects, the amino acid substitutions areconservative amino acid substitutions. In some embodiments, the ABPsdescribed in this paragraph are referred to herein as “variants.” Insome embodiments, such variants are derived from a sequence providedherein, for example, by affinity maturation, site directed mutagenesis,random mutagenesis, or any other method known in the art or describedherein. In some embodiments, such variants are not derived from asequence provided herein and may, for example, be isolated de novoaccording to the methods provided herein for obtaining ABPs.

In some embodiments, an ABP provided herein comprises a CDR-H3 selectedfrom SEQ ID NOs: 29-35 and a CDR-H2 selected from SEQ ID NOs: 36-47. Insome embodiments, an ABP provided herein comprises a CDR-H3 selectedfrom SEQ ID NOs: 29-35, a CDR-H2 selected from SEQ ID NOs: 36-47, and aCDR-H1 selected from SEQ ID NOs: 48-54 or 58-62. In some embodiments,the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identitywith a CDR-H3 of SEQ ID NOs: 29-35, the CDR-H2 has at least about 50%,75%, 80%, 85%, 90%, or 95% identity with a CDR-H2 of SEQ ID NOs: 36-47,and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95%identity with a CDR-H1 of SEQ ID NOs: 48-54 or 58-62. In someembodiments, the CDR-H3 is a CDR-H3 selected from SEQ ID NOs: 29-35,with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; theCDR-H2 is a CDR-H2 selected from SEQ ID NOs: 36-47, with up to 1, 2, 3,4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is a CDR-H1selected from SEQ ID NOs: 48-54 or 58-62, with up to 1, 2, 3, 4, or 5amino acid substitutions. In some aspects, the amino acid substitutionsare conservative amino acid substitutions. In some embodiments, the ABPsdescribed in this paragraph are referred to herein as “variants.” Insome embodiments, such variants are derived from a sequence providedherein, for example, by affinity maturation, site directed mutagenesis,random mutagenesis, or any other method known in the art or describedherein. In some embodiments, such variants are not derived from asequence provided herein and may, for example, be isolated de novoaccording to the methods provided herein for obtaining ABPs.

In some embodiments, an ABP provided herein comprises a CDR-L3 selectedfrom SEQ ID NOs: 63-66. In some aspects, the CDR-L3 has at least about50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L3 of SEQ ID NOs:63-66. In some aspects, the CDR-L3 is a CDR-L3 according to the Kabat,Chothia, and IMGT numbering systems. In some embodiments, the CDR-L3 isa CDR-L3 selected from SEQ ID NOs: 63-66, with up to 1, 2, 3, 4, or 5amino acid substitutions. In some aspects, the amino acid substitutionsare conservative amino acid substitutions. In some embodiments, the ABPsdescribed in this paragraph are referred to herein as “variants.” Insome embodiments, such variants are derived from a sequence providedherein, for example, by affinity maturation, site directed mutagenesis,random mutagenesis, or any other method known in the art or describedherein. In some embodiments, such variants are not derived from asequence provided herein and may, for example, be isolated de novoaccording to the methods provided herein for obtaining ABPs.

In some embodiments, an ABP provided herein comprises a CDR-L2 selectedfrom SEQ ID NOs: 67-69. In some aspects, the CDR-L2 has at least about50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L2 of SEQ ID NOs:67-69. In some aspects, the CDR-L2 is a CDR-L2 according to the Kabatand Chothia numbering systems. In some embodiments, the CDR-L2 is aCDR-L2 selected from SEQ ID NOs: 67-69, with up to 1, 2, 3, or 4 aminoacid substitutions. In some aspects, the amino acid substitutions areconservative amino acid substitutions. In some embodiments, the ABPsdescribed in this paragraph are referred to herein as “variants.” Insome embodiments, such variants are derived from a sequence providedherein, for example, by affinity maturation, site directed mutagenesis,random mutagenesis, or any other method known in the art or describedherein. In some embodiments, such variants are not derived from asequence provided herein and may, for example, be isolated de novoaccording to the methods provided herein for obtaining ABPs.

In some embodiments, an ABP provided herein comprises a CDR-L1 selectedfrom SEQ ID NOs: 70-72. In some aspects, the CDR-L1 has at least about50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L1 of SEQ ID NOs:70-72. In some aspects, the CDR-L1 is a CDR-L1 according to the Kabatand Chothia numbering systems. In some embodiments, the CDR-L1 is aCDR-L1 selected from SEQ ID NOs: 70-72, with up to 1, 2, 3, 4, 5, or 6amino acid substitutions. In some aspects, the amino acid substitutionsare conservative amino acid substitutions. In some embodiments, the ABPsdescribed in this paragraph are referred to herein as “variants.” Insome embodiments, such variants are derived from a sequence providedherein, for example, by affinity maturation, site directed mutagenesis,random mutagenesis, or any other method known in the art or describedherein. In some embodiments, such variants are not derived from asequence provided herein and may, for example, be isolated de novoaccording to the methods provided herein for obtaining ABPs.

In some embodiments, an ABP provided herein comprises a CDR-L3 selectedfrom SEQ ID NOs: 63-66 and a CDR-L2 selected from SEQ ID NOs: 67-69. Insome embodiments, an ABP provided herein comprises a CDR-L3 selectedfrom SEQ ID NOs: 63-66, a CDR-L2 selected from SEQ ID NOs: 67-69, and aCDR-L1 selected from SEQ ID NOs: 70-72. In some embodiments, the CDR-L3has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with aCDR-L3 of SEQ ID NOs: 63-66, the CDR-L2 has at least about 50%, 75%,80%, 85%, 90%, or 95% identity with a CDR-L2 of SEQ ID NOs: 67-69, andthe CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identitywith a CDR-L1 of SEQ ID NOs: 70-72. In some embodiments, the CDR-L3 is aCDR-L3 selected from SEQ ID NOs: 63-66, with up to 1, 2, 3, 4, or 5amino acid substitutions; the CDR-L2 is a CDR-L2 selected from SEQ IDNOs: 67-69, with up to 1, 2, 3, or 4 amino acid substitutions; and theCDR-L1 is a CDR-L1 selected from SEQ ID NOs: 70-72, with up to 1, 2, 3,4, 5, or 6 amino acid substitutions. In some aspects, the amino acidsubstitutions are conservative amino acid substitutions. In someembodiments, the ABPs described in this paragraph are referred to hereinas “variants.” In some embodiments, such variants are derived from asequence provided herein, for example, by affinity maturation, sitedirected mutagenesis, random mutagenesis, or any other method known inthe art or described herein. In some embodiments, such variants are notderived from a sequence provided herein and may, for example, beisolated de novo according to the methods provided herein for obtainingABPs.

In some embodiments, an ABP provided herein comprises a CDR-H3 selectedfrom SEQ ID NOs: 29-35, a CDR-H2 selected from SEQ ID NOs: 36-47, aCDR-H1 selected from SEQ ID NOs: 48-54 or 58-62, a CDR-L3 selected fromSEQ ID NOs: 63-66, a CDR-L2 selected from SEQ ID NOs: 67-69, and aCDR-L1 selected from SEQ ID NOs: 70-72. In some embodiments, the CDR-H3has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with aCDR-H3 of SEQ ID NOs: 29-35, the CDR-H2 has at least about 50%, 75%,80%, 85%, 90%, or 95% identity with a CDR-H2 of SEQ ID NOs: 36-47, theCDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity witha CDR-H1 of SEQ ID NOs: 48-54 or 58-62, the CDR-L3 has at least about50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L3 of SEQ ID NOs:63-66, the CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95%identity with a CDR-L2 of SEQ ID NOs: 67-69, and the CDR-L1 has at leastabout 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L1 of SEQ IDNOs: 70-72. In some embodiments, the CDR-H3 is a CDR-H3 selected fromSEQ ID NOs: 29-35, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acidsubstitutions; the CDR-H2 is a CDR-H2 selected from SEQ ID NOs: 36-47,with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; theCDR-H1 is a CDR-H1 selected from SEQ ID NOs: 48-54 or 58-62, with up to1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is a CDR-L3selected from SEQ ID NOs: 63-66, with up to 1, 2, 3, 4, or 5 amino acidsubstitutions; the CDR-L2 is a CDR-L2 selected from SEQ ID NOs: 67-69,with up to 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is aCDR-L1 selected from SEQ ID NOs: 70-72, with up to 1, 2, 3, 4, 5, or 6amino acid substitutions. In some aspects, the amino acid substitutionsare conservative amino acid substitutions. In some embodiments, the ABPsdescribed in this paragraph are referred to herein as “variants.” Insome embodiments, such variants are derived from a sequence providedherein, for example, by affinity maturation, site directed mutagenesis,random mutagenesis, or any other method known in the art or describedherein. In some embodiments, such variants are not derived from asequence provided herein and may, for example, be isolated de novoaccording to the methods provided herein for obtaining ABPs.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:29, a CDR-H2 of SEQ ID NO:36, a CDR-H1 of SEQ ID NO:48, a CDR-L3 ofSEQ ID NO:63, a CDR-L2 of SEQ ID NO:67, and a CDR-L1 of SEQ ID NO:70.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:29, a CDR-H2 of SEQ ID NO:37, a CDR-H1 of SEQ ID NO:49, a CDR-L3 ofSEQ ID NO:63, a CDR-L2 of SEQ ID NO:67, and a CDR-L1 of SEQ ID NO:70.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:29, a CDR-H2 of SEQ ID NO:37, a CDR-H1 of SEQ ID NO:50, a CDR-L3 ofSEQ ID NO:63, a CDR-L2 of SEQ ID NO:67, and a CDR-L1 of SEQ ID NO:70.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:30, a CDR-H2 of SEQ ID NO:37, a CDR-H1 of SEQ ID NO:50, a CDR-L3 ofSEQ ID NO:63, a CDR-L2 of SEQ ID NO:67, and a CDR-L1 of SEQ ID NO:70.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:29, a CDR-H2 of SEQ ID NO:38, a CDR-H1 of SEQ ID NO:50, a CDR-L3 ofSEQ ID NO:63, a CDR-L2 of SEQ ID NO:67, and a CDR-L1 of SEQ ID NO:70.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:31, a CDR-H2 of SEQ ID NO:39, a CDR-H1 of SEQ ID NO:51, a CDR-L3 ofSEQ ID NO:64, a CDR-L2 of SEQ ID NO:68, and a CDR-L1 of SEQ ID NO:71.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:31, a CDR-H2 of SEQ ID NO:40, a CDR-H1 of SEQ ID NO:52, a CDR-L3 ofSEQ ID NO:64, a CDR-L2 of SEQ ID NO:68, and a CDR-L1 of SEQ ID NO:71.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:31, a CDR-H2 of SEQ ID NO:41, a CDR-H1 of SEQ ID NO:53, a CDR-L3 ofSEQ ID NO:64, a CDR-L2 of SEQ ID NO:68, and a CDR-L1 of SEQ ID NO:71.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:31, a CDR-H2 of SEQ ID NO:40, a CDR-H1 of SEQ ID NO:54, a CDR-L3 ofSEQ ID NO:64, a CDR-L2 of SEQ ID NO:68, and a CDR-L1 of SEQ ID NO:71.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:32, a CDR-H2 of SEQ ID NO:40, a CDR-H1 of SEQ ID NO:54, a CDR-L3 ofSEQ ID NO:64, a CDR-L2 of SEQ ID NO:68, and a CDR-L1 of SEQ ID NO:71.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:32, a CDR-H2 of SEQ ID NO:40, a CDR-H1 of SEQ ID NO:54, a CDR-L3 ofSEQ ID NO:64, a CDR-L2 of SEQ ID NO:68, and a CDR-L1 of SEQ ID NO:71.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:32, a CDR-H2 of SEQ ID NO:40, a CDR-H1 of SEQ ID NO:54, a CDR-L3 ofSEQ ID NO:64, a CDR-L2 of SEQ ID NO:68, and a CDR-L1 of SEQ ID NO:71.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:33, a CDR-H2 of SEQ ID NO:42, a CDR-H1 of SEQ ID NO:58, a CDR-L3 ofSEQ ID NO:65, a CDR-L2 of SEQ ID NO:69, and a CDR-L1 of SEQ ID NO:72.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:33, a CDR-H2 of SEQ ID NO:42, a CDR-H1 of SEQ ID NO:59, a CDR-L3 ofSEQ ID NO:65, a CDR-L2 of SEQ ID NO:69, and a CDR-L1 of SEQ ID NO:72.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:33, a CDR-H2 of SEQ ID NO:43, a CDR-H1 of SEQ ID NO:60, a CDR-L3 ofSEQ ID NO:65, a CDR-L2 of SEQ ID NO:69, and a CDR-L1 of SEQ ID NO:72.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:34, a CDR-H2 of SEQ ID NO:43, a CDR-H1 of SEQ ID NO:60, a CDR-L3 ofSEQ ID NO:65, a CDR-L2 of SEQ ID NO:69, and a CDR-L1 of SEQ ID NO:72.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:34, a CDR-H2 of SEQ ID NO:44, a CDR-H1 of SEQ ID NO:61, a CDR-L3 ofSEQ ID NO:65, a CDR-L2 of SEQ ID NO:69, and a CDR-L1 of SEQ ID NO:72.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:33, a CDR-H2 of SEQ ID NO:44, a CDR-H1 of SEQ ID NO:59, a CDR-L3 ofSEQ ID NO:65, a CDR-L2 of SEQ ID NO:69, and a CDR-L1 of SEQ ID NO:72.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:35, a CDR-H2 of SEQ ID NO:45, a CDR-H1 of SEQ ID NO:62, a CDR-L3 ofSEQ ID NO:66, a CDR-L2 of SEQ ID NO:69, and a CDR-L1 of SEQ ID NO:72.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:35, a CDR-H2 of SEQ ID NO:46, a CDR-H1 of SEQ ID NO:62, a CDR-L3 ofSEQ ID NO:66, a CDR-L2 of SEQ ID NO:69, and a CDR-L1 of SEQ ID NO:72.

In some embodiments, an ABP provided herein comprises a CDR-H3 of SEQ IDNO:35, a CDR-H2 of SEQ ID NO:47, a CDR-H1 of SEQ ID NO:62, a CDR-L3 ofSEQ ID NO:66, a CDR-L2 of SEQ ID NO:69, and a CDR-L1 of SEQ ID NO:72.

2.2.5. Heavy Chains and Light Chains

In some embodiments, an ABP provided herein comprises a V_(H) selectedfrom a V_(H) of SEQ ID NO:4-24 (or a variant described herein) and aconstant region selected from SEQ ID NOs: 55-57 or 125. In someembodiments, an ABP provided herein comprises a V_(L) selected from aV_(L) of SEQ ID NO:25-28 (or a variant described herein) and a constantregion of SEQ ID NO:126.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:79. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:80. In some embodiments, an ABP provided hereincomprises a light chain of SEQ ID NO:81. In some embodiments, an APBprovided herein comprises a heavy chain of SEQ ID NO:79 and a lightchain of SEQ ID NO: 81. In some embodiments, an APB provided hereincomprises a heavy chain of SEQ ID NO:80 and a light chain of SEQ ID NO:81.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:82. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:83. In some embodiments, an APB provided hereincomprises a heavy chain of SEQ ID NO:82 and a light chain of SEQ ID NO:81. In some embodiments, an APB provided herein comprises a heavy chainof SEQ ID NO:83 and a light chain of SEQ ID NO: 81.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:84. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:85. In some embodiments, an APB provided hereincomprises a heavy chain of SEQ ID NO:84 and a light chain of SEQ ID NO:81. In some embodiments, an APB provided herein comprises a heavy chainof SEQ ID NO:85 and a light chain of SEQ ID NO: 81.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:86. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:87. In some embodiments, an APB provided hereincomprises a heavy chain of SEQ ID NO:86 and a light chain of SEQ ID NO:81. In some embodiments, an APB provided herein comprises a heavy chainof SEQ ID NO:87 and a light chain of SEQ ID NO: 81.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:88. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:89. In some embodiments, an APB provided hereincomprises a heavy chain of SEQ ID NO:88 and a light chain of SEQ ID NO:81. In some embodiments, an APB provided herein comprises a heavy chainof SEQ ID NO:89 and a light chain of SEQ ID NO: 81.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:90. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:91. In some embodiments, an ABP provided hereincomprises a light chain of SEQ ID NO:92. In some embodiments, an APBprovided herein comprises a heavy chain of SEQ ID NO:90 and a lightchain of SEQ ID NO:92. In some embodiments, an APB provided hereincomprises a heavy chain of SEQ ID NO:91 and a light chain of SEQ IDNO:92.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:93. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:94. In some embodiments, an APB provided hereincomprises a heavy chain of SEQ ID NO:93 and a light chain of SEQ IDNO:92. In some embodiments, an APB provided herein comprises a heavychain of SEQ ID NO:94 and a light chain of SEQ ID NO:92.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:95. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:96. In some embodiments, an APB provided hereincomprises a heavy chain of SEQ ID NO:95 and a light chain of SEQ IDNO:92. In some embodiments, an APB provided herein comprises a heavychain of SEQ ID NO:96 and a light chain of SEQ ID NO:92.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:97. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:98. In some embodiments, an APB provided hereincomprises a heavy chain of SEQ ID NO:97 and a light chain of SEQ IDNO:92. In some embodiments, an APB provided herein comprises a heavychain of SEQ ID NO:98 and a light chain of SEQ ID NO:92.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:99. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:100. In some embodiments, an APB providedherein comprises a heavy chain of SEQ ID NO:99 and a light chain of SEQID NO:92. In some embodiments, an APB provided herein comprises a heavychain of SEQ ID NO:100 and a light chain of SEQ ID NO:92.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:101. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:102. In some embodiments, an APB providedherein comprises a heavy chain of SEQ ID NO:101 and a light chain of SEQID NO:92. In some embodiments, an APB provided herein comprises a heavychain of SEQ ID NO:102 and a light chain of SEQ ID NO:92.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:103. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:104. In some embodiments, an APB providedherein comprises a heavy chain of SEQ ID NO:103 and a light chain of SEQID NO:92. In some embodiments, an APB provided herein comprises a heavychain of SEQ ID NO:104 and a light chain of SEQ ID NO:92.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:105. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:106. In some embodiments, an ABP providedherein comprises a light chain of SEQ ID NO:107. In some embodiments, anAPB provided herein comprises a heavy chain of SEQ ID NO:105 and a lightchain of SEQ ID NO:107. In some embodiments, an APB provided hereincomprises a heavy chain of SEQ ID NO:106 and a light chain of SEQ IDNO:107.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:108. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:109. In some embodiments, an APB providedherein comprises a heavy chain of SEQ ID NO:108 and a light chain of SEQID NO:107. In some embodiments, an APB provided herein comprises a heavychain of SEQ ID NO:109 and a light chain of SEQ ID NO:107.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:110. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:111. In some embodiments, an APB providedherein comprises a heavy chain of SEQ ID NO:110 and a light chain of SEQID NO:107. In some embodiments, an APB provided herein comprises a heavychain of SEQ ID NO:111 and a light chain of SEQ ID NO:107.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:112. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:113. In some embodiments, an APB providedherein comprises a heavy chain of SEQ ID NO:112 and a light chain of SEQID NO:107. In some embodiments, an APB provided herein comprises a heavychain of SEQ ID NO:113 and a light chain of SEQ ID NO:107.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:114. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:115. In some embodiments, an APB providedherein comprises a heavy chain of SEQ ID NO:114 and a light chain of SEQID NO:107. In some embodiments, an APB provided herein comprises a heavychain of SEQ ID NO:115 and a light chain of SEQ ID NO:107.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:116. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:117. In some embodiments, an APB providedherein comprises a heavy chain of SEQ ID NO:116 and a light chain of SEQID NO:107. In some embodiments, an APB provided herein comprises a heavychain of SEQ ID NO:117 and a light chain of SEQ ID NO:107.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:118. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:119. In some embodiments, an ABP providedherein comprises a light chain of SEQ ID NO:120. In some embodiments, anAPB provided herein comprises a heavy chain of SEQ ID NO:118 and a lightchain of SEQ ID NO:120. In some embodiments, an APB provided hereincomprises a heavy chain of SEQ ID NO:119 and a light chain of SEQ IDNO:120.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:121. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:122. In some embodiments, an APB providedherein comprises a heavy chain of SEQ ID NO:121 and a light chain of SEQID NO:120. In some embodiments, an APB provided herein comprises a heavychain of SEQ ID NO:122 and a light chain of SEQ ID NO:120.

In some embodiments, an ABP provided herein comprises a heavy chain ofSEQ ID NO:123. In some embodiments, an ABP provided herein comprises aheavy chain of SEQ ID NO:124. In some embodiments, an APB providedherein comprises a heavy chain of SEQ ID NO:123 and a light chain of SEQID NO:120. In some embodiments, an APB provided herein comprises a heavychain of SEQ ID NO:124 and a light chain of SEQ ID NO:120.

2.2.6. Consensus Sequences

In some embodiments, provided herein is a first family of ABPs, whereinan ABP of such family comprises the following six CDR sequences: (a) aCDR-H3 having the sequence A-R-D-G-V-L-X₁-L-N-K-R-S-F-D-I, wherein X₁ isA or T (SEQ ID NO: 128); (b) a CDR-H2 having the sequenceS-I-Y-Y-S-G-X₂-T-Y-Y-N-P-S-L-K-S, wherein X₂ is 5, Q or G (SEQ ID NO:129); (c) a CDR-H1 having the sequence G-S-I-X₃-S-G-X₄-Y—Y-W-G, whereinX₃ is E or A, and X₄ is L, V or S (SEQ ID NO: 130); (d) a CDR-L3 havingthe sequence QQHTVRPPLT (SEQ ID NO: 64); (e) a CDR-L2 having thesequence GASSRAT (SEQ ID NO: 68); and (f) a CDR-L1 having the sequenceRASQSVSSSYLA (SEQ ID NO: 71). In some embodiments, provided herein is anABP within such first family.

In some embodiments, provided herein is a second family of ABPs, whereinan ABP of such family comprises the following six CDR sequences: (a) aCDR-H3 having the sequence A-R-D-A-N—Y-Y-G-X₁-A-W-A-F-D-P, wherein X₁ isS or G (SEQ ID NO: 131); (b) a CDR-H2 having the sequenceS—I-Y-Y-S-G-X₂-T-F-Y-N-P-S-L-K-X₃, wherein X₂ is S or A, and X₃ is S orG (SEQ ID NO: 132); (c) a CDR-H1 having the sequenceG-S-I-X₄-S-X₅-X₆-X₇-Y-W-G, wherein X₄ is S or T, X₅ is S or T, X₆ is Sor K, and X₇ is H or Y (SEQ ID NO: 133); (d) a CDR-L3 having thesequence QQHFNLPT (SEQ ID NO: 63); (e) a CDR-L2 having the sequenceDASNRAT (SEQ ID NO: 67); and (f) a CDR-L1 having the sequenceRASQSVSSYLA (SEQ ID NO: 70). In some embodiments, provided herein is anABP within such second family.

In some embodiments, provided herein is a third family of ABPs, whereinan ABP of such family comprises the following six CDR sequences: (a) aCDR-H3 having the sequence A-R-G-G-R-T-T-W-I-G-A-X₁-D-I, wherein X₁ is For L (SEQ ID NO: 134); (b) a CDR-H2 having the sequenceI—I-N-P-S-X₂-G-L-T-S-Y-A-X₃-K-F-Q-G, wherein X₂ is L or I, and X₃ is Qor R (SEQ ID NO: 135); (c) a CDR-H1 having the sequenceY-T-F-X₄-X₅-Y-Y-X₆-H, wherein X₄ is G, P or R, X₅ is N, A or E, and X₆is M or I (SEQ ID NO: 136); (d) a CDR-L3 having the sequence QQYVVWPPLT(SEQ ID NO:65); (e) a CDR-L2 having the sequence GASTRAT (SEQ ID NO:69);and (f) a CDR-L1 having the sequence RASQSVSSNLA (SEQ ID NO:72). In someembodiments, provided herein is an ABP within such third family.

In some embodiments, provided herein is a fourth family of ABPs, whereinan ABP of such family comprises the following six CDR sequences: (a) aCDR-H3 having the sequence ARLHVSGSYYPAYLDY (SEQ ID NO: 35); (b) aCDR-H2 having the sequence X₁-I-N-P-S-M-G-A-T-S—Y-X₂-Q-K-F-X₃-G, whereinX₁ is V or I, X₂ is A or T, and X₃ is Q or R (SEQ ID NO: 137); (c) aCDR-H1 having the sequence YTFTSHYMG (SEQ ID NO: 62); (d) a CDR-L3having the sequence QQYIVFPWT (SEQ ID NO: 66); (e) a CDR-L2 having thesequence GASTRAT (SEQ ID NO: 69); and (f) a CDR-L1 having the sequenceRASQSVSSNLA, (SEQ ID NO: 72). In some embodiments, provided herein is anABP within such fourth family.

2.2.7. Functional Properties of ABP Variants

As described above, and elsewhere in this disclosure, provided hereinare ABP variants defined based on percent identity to an illustrativeABP sequence provided herein, or substitution of amino acid residues incomparison to an illustrative ABP sequence provided herein.

In some embodiments, a variant of an ABP provided herein has specificityfor hTIGIT. In some embodiments, a variant of an ABP provided herein hasspecificity for cTIGIT. In some embodiments, a variant of an ABPprovided herein has specificity for mTIGIT. In some embodiments, avariant of an ABP provided herein has specificity for hTIGIT and cTIGIT.In some embodiments, a variant of an ABP provided herein has specificityfor hTIGIT and mTIGIT. In some embodiments, a variant of an ABP providedherein has specificity for cTIGIT and mTIGIT. In some embodiments, avariant of an ABP provided herein has specificity for hTIGIT, cTIGIT andmTIGIT.

In some embodiments, a variant of an ABP that is derived from anillustrative ABP sequence provided herein retains affinity, as measuredby K_(D), for hTIGIT that is within about 1.5-fold, about 2-fold, about3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about8-fold, about 9-fold or about 10-fold the affinity of such illustrativeABP. In some embodiments, a variant of an ABP that is derived from anillustrative ABP sequence provided herein retains affinity, as measuredby K_(D), for cTIGIT that is within about 1.5-fold, about 2-fold, about3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about8-fold, about 9-fold or about 10-fold the affinity of such illustrativeABP. In some embodiments, a variant of an ABP that is derived from anillustrative ABP sequence provided herein retains affinity, as measuredby K_(D), for mTIGIT that is within about 1.5-fold, about 2-fold, about3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about8-fold, about 9-fold or about 10-fold the affinity of such illustrativeABP. In some embodiments, a variant of an ABP that is derived from anillustrative ABP sequence provided herein retains affinity, as measuredby K_(D), for both hTIGIT and cTIGIT that is within about 1.5-fold,about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold,about 7-fold, about 8-fold, about 9-fold or about 10-fold the affinityof such illustrative ABP. In some embodiments, a variant of an ABP thatis derived from an illustrative ABP sequence provided herein retainsaffinity, as measured by K_(D), for both hTIGIT and mTIGIT that iswithin about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold or about10-fold the affinity of such illustrative ABP. In some embodiments, avariant of an ABP that is derived from an illustrative ABP sequenceprovided herein retains affinity, as measured by K_(D), for both cTIGITand mTIGIT that is within about 1.5-fold, about 2-fold, about 3-fold,about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold,about 9-fold or about 10-fold the affinity of such illustrative ABP. Insome embodiments, a variant of an ABP that is derived from anillustrative ABP sequence provided herein retains affinity, as measuredby K_(D), for all three of hTIGIT, cTIGIT and mTIGIT that is withinabout 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold,about 6-fold, about 7-fold, about 8-fold, about 9-fold or about 10-foldthe affinity of such illustrative ABP.

In some embodiments, a variant of an ABP provided herein retains theability to antagonize TIGIT, as measured by one or more assays orbiological effects described herein. In some embodiments, a variant ofan ABP provided herein retains the ability to prevent TIGIT frominteracting with one or more of its ligands, as described herein.

In some embodiments, a variant of an ABP provided herein competes forbinding to TIGIT with an antibody selected from MAB1, MAB2, MAB3, MAB4,MAB5, MAB6, MAB7, MAB8, MAB9, MAB10, MAB11, MAB12, MAB13, MAB14, MAB15,MAB16, MAB17, MAB18, MAB19, MAB20, or MAB21, each as provided in Table 5of this disclosure.

In some embodiments, a variant of an ABP provided herein inhibitsbinding of CD155 to TIGIT. In some embodiments, a variant of an ABPprovided herein inhibits binding of CD112 to TIGIT. In some embodiments,a variant of an ABP provided herein inhibits association of CD226 withTIGIT.

In some embodiments, a variant of an ABP provided herein activates aneffector T cell or a natural killer (NK) cell. In some embodiments, avariant of an ABP provided herein decreases the number of regulatory Tcells in a tissue or in circulation. In some embodiments, a variant ofan ABP provided herein inhibits the suppression of an effector T cell bya regulatory T cell.

In some embodiments, a variant of an ABP provided herein does not bindspecifically to any of PVRL1, PVRL2, PVRL3, or PVRL4.

In some embodiments, a variant of an ABP provided herein binds murineTIGIT (SEQ ID NO: 3) with an affinity lower (as indicated by higherK_(D)) than the affinity of the ABP for hTIGIT, or does not bind mTIGIT.In some embodiments, a variant of an ABP provided herein binds murineTIGIT (SEQ ID NO: 138) with an affinity lower (as indicated by higherK_(D)) than the affinity of the ABP for hTIGIT, or does not bind mTIGIT.

In some embodiments, a variant of an ABP provided herein binds the sameepitope of TIGIT as such ABP.

2.2.8. Other Functional Properties of ABPs

In some embodiments, an ABP provided herein has one or more of thecharacteristics listed in the following (a)-(j): (a) competes forbinding to TIGIT with an antibody selected from MAB1, MAB2, MAB3, MAB4,MAB5, MAB6, MAB7, MAB8, MAB9, MAB10, MAB11, MAB12, MAB13, MAB14, MAB15,MAB16, MAB17, MAB18, MAB19, MAB20, or MAB21, each as provided in Table 5of this disclosure; (b) inhibits binding of CD155 to TIGIT; (c) inhibitsbinding of CD112 to TIGIT; (d) inhibits association of CD226 with TIGIT;(e) activates an effector T cell or a natural killer (NK) cell; (f)decreases the number of regulatory T cells in a tissue or incirculation; (g) inhibits the suppression of an effector T cell by aregulatory T cell; (h) does not bind specifically to any of PVRL1,PVRL2, PVRL3, or PVRL4; (i) specifically binds cynomolgus monkey TIGIT(cTIGIT; SEQ ID NO: 2); or (j) binds murine TIGIT (mTIGIT; SEQ ID NO: 3)with an affinity lower (as indicated by higher K_(D)) than the affinityof the ABP for hTIGIT, or does not bind mTIGIT. In some embodiments, anABP provided herein has two or more of the characteristics listed in theforegoing (a)-(j). In some embodiments, an ABP provided herein has threeor more of the characteristics listed in the foregoing (a)-(j). In someembodiments, an ABP provided herein has four or more of thecharacteristics listed in the foregoing (a)-(j). In some embodiments, anABP provided herein has five or more of the characteristics listed inthe foregoing (a)-(j). In some embodiments, an ABP provided herein hassix or more of the characteristics listed in the foregoing (a)-(j). Insome embodiments, an ABP provided herein has seven or more of thecharacteristics listed in the foregoing (a)-(j). In some embodiments, anABP provided herein has eight or more of the characteristics listed inthe foregoing (a)-(j). In some embodiments, an ABP provided herein hasnine or more of the characteristics listed in the foregoing (a)-(j). Insome embodiments, an ABP provided herein has all ten of thecharacteristics listed in the foregoing (a)-(j).

In some embodiments, an ABP provided herein exhibits a combination ofthe characteristics listed in the following (a)-(j): (a) competes forbinding to TIGIT with an antibody selected from MAB1, MAB2, MAB3, MAB4,MAB5, MAB6, MAB7, MAB8, MAB9, MAB10, MAB11, MAB12, MAB13, MAB14, MAB15,MAB16, MAB17, MAB18, MAB19, MAB20, or MAB21, each as provided in Table 5of this disclosure; (b) inhibits binding of CD155 to TIGIT; (c) inhibitsbinding of CD112 to TIGIT; (d) inhibits association of CD226 with TIGIT;(e) activates an effector T cell or a natural killer (NK) cell; (f)decreases the number of regulatory T cells in a tissue or incirculation; (g) inhibits the suppression of an effector T cell by aregulatory T cell; (h) does not bind specifically to any of PVRL1,PVRL2, PVRL3, or PVRL4; (i) specifically binds cynomolgus monkey TIGIT(cTIGIT; SEQ ID NO: 2); or (j) binds murine TIGIT (mTIGIT; SEQ ID NO: 3)with an affinity lower (as indicated by higher K_(D)) than the affinityof the ABP for hTIGIT, or does not bind mTIGIT. In some embodiments,such ABP exhibits a combination of the characteristics selected from (aand b), (a and c), (a and d), (a and e), (a and f), (a and g), (a andh), (a and i), (a and j), (b and a), (b and c), (b and d), (b and e), (band f), (b and g), (b and h), (b and i), (b and j), (c and a), (c andb), (c and d), (c and e), (c and f), (c and g), (c and h), (c and i), (cand j), (d and a), (d and b), (d and c), (d and e), (d and f), (d andg), (d and h), (d and i), (d and j), (e and a), (e and b), (e and c), (eand d), (e and f), (e and g), (e and h), (e and i), (e and j), (f anda), (f and b), (f and c), (f and d), (f and e), (f and g), (f and h), (fand i), (f and j), (g and a), (g and b), (g and c), (g and d), (g ande), (g and f), (g and h), (g and i), (g and j), (h and a), (h and b), (hand c), (h and d), (h and e), (h and f), (h and g), (h and i), (h andj), (i and a), (i and b), (i and c), (i and d), (i and e), (i and f), (iand g), (i and h), (i and j), (j and a), (j and b), (j and c), (j andd), (j and e), (j and f), (j and g), (j and h), and (j and i). In someembodiments, such ABP exhibits a combination of the characteristicsselected from (a and b and c), (a and b and d), (a and b and e), (a andb and f), (a and b and g), (a and b and h), (a and b and i), (a and band j), (a and c and b), (a and c and d), (a and c and e), (a and c andf), (a and c and g), (a and c and h), (a and c and i), (a and c and j),(a and d and b), (a and d and c), (a and d and e), (a and d and f), (aand d and g), (a and d and h), (a and d and i), (a and d and j), (a ande and b), (a and e and c), (a and e and d), (a and e and f), (a and eand g), (a and e and h), (a and e and i), (a and e and j), (a and f andb), (a and f and c), (a and f and d), (a and f and e), (a and f and g),(a and f and h), (a and f and i), (a and f and j), (a and g and b), (aand g and c), (a and g and d), (a and g and e), (a and g and f), (a andg and h), (a and g and i), (a and g and j), (a and h and b), (a and hand c), (a and h and d), (a and h and e), (a and h and f), (a and h andg), (a and h and i), (a and h and j), (a and i and b), (a and i and c),(a and i and d), (a and i and e), (a and i and f), (a and i and g), (aand i and h), (a and i and j), (a and j and b), (a and j and c), (a andj and d), (a and j and e), (a and j and f), (a and j and g), (a and jand h), (a and j and i), (b and a and j), (b and a and c), (b and a andd), (b and a and e), (b and a and f), (b and a and g), (b and a and h),(b and a and i), (b and c and j), (b and c and a), (b and c and d), (band c and e), (b and c and f), (b and c and g), (b and c and h), (b andc and i), (b and d and j), (b and d and a), (b and d and c), (b and dand e), (b and d and f), (b and d and g), (b and d and h), (b and d andi), (b and e and j), (b and e and a), (b and e and c), (b and e and d),(b and e and f), (b and e and g), (b and e and h), (b and e and i), (band f and j), (b and f and a), (b and f and c), (b and f and d), (b andf and e), (b and f and g), (b and f and h), (b and f and i), (b and gand j), (b and g and a), (b and g and c), (b and g and d), (b and g ande), (b and g and f), (b and g and h), (b and g and i), (b and h and j),(b and h and a), (b and h and c), (b and h and d), (b and h and e), (band h and f), (b and h and g), (b and h and i), (b and i and j), (b andi and a), (b and i and c), (b and i and d), (b and i and e), (b and iand f), (b and i and g), (b and i and h), (b and j and i), (b and j anda), (b and j and c), (b and j and d), (b and j and e), (b and j and f),(b and j and g), (b and j and h), (c and a and i), (c and a and j), (cand a and b), (c and a and d), (c and a and e), (c and a and f), (c anda and g), (c and a and h), (c and b and i), (c and b and j), (c and band a), (c and b and d), (c and b and e), (c and b and f), (c and b andg), (c and b and h), (c and d and i), (c and d and j), (c and d and a),(c and d and b), (c and d and e), (c and d and f), (c and d and g), (cand d and h), (c and e and i), (c and e and j), (c and e and a), (c ande and b), (c and e and d), (c and e and f), (c and e and g), (c and eand h), (c and f and i), (c and f and j), (c and f and a), (c and f andb), (c and f and d), (c and f and e), (c and f and g), (c and f and h),(c and g and i), (c and g and j), (c and g and a), (c and g and b), (cand g and d), (c and g and e), (c and g and f), (c and g and h), (c andh and i), (c and h and j), (c and h and a), (c and h and b), (c and hand d), (c and h and e), (c and h and f), (c and h and g), (c and i andh), (c and i and j), (c and i and a), (c and i and b), (c and i and d),(c and i and e), (c and i and f), (c and i and g), (c and j and h), (cand j and i), (c and j and a), (c and j and b), (c and j and d), (c andj and e), (c and j and f), (c and j and g), (d and a and h), (d and aand i), (d and a and j), (d and a and b), (d and a and c), (d and a ande), (d and a and f), (d and a and g), (d and b and h), (d and b and i),(d and b and j), (d and b and a), (d and b and c), (d and b and e), (dand b and f), (d and b and g), (d and c and h), (d and c and i), (d andc and j), (d and c and a), (d and c and b), (d and c and e), (d and cand f), (d and c and g), (d and e and h), (d and e and i), (d and e andj), (d and e and a), (d and e and b), (d and e and c), (d and e and f),(d and e and g), (d and f and h), (d and f and i), (d and f and j), (dand f and a), (d and f and b), (d and f and c), (d and f and e), (d andf and g), (d and g and h), (d and g and i), (d and g and j), (d and gand a), (d and g and b), (d and g and c), (d and g and e), (d and g andf), (d and h and g), (d and h and i), (d and h and j), (d and h and a),(d and h and b), (d and h and c), (d and h and e), (d and h and f), (dand i and g), (d and i and h), (d and i and j), (d and i and a), (d andi and b), (d and i and c), (d and i and e), (d and i and f), (d and jand g), (d and j and h), (d and j and i), (d and j and a), (d and j andb), (d and j and c), (d and j and e), (d and j and f), (e and a and g),(e and a and h), (e and a and i), (e and a and j), (e and a and b), (eand a and c), (e and a and d), (e and a and f), (e and b and g), (e andb and h), (e and b and i), (e and b and j), (e and b and a), (e and band c), (e and b and d), (e and b and f), (e and c and g), (e and c andh), (e and c and i), (e and c and j), (e and c and a), (e and c and b),(e and c and d), (e and c and f), (e and d and g), (e and d and h), (eand d and i), (e and d and j), (e and d and a), (e and d and b), (e andd and c), (e and d and f), (e and f and g), (e and f and h), (e and fand i), (e and f and j), (e and f and a), (e and f and b), (e and f andc), (e and f and d), (e and g and f), (e and g and h), (e and g and i),(e and g and j), (e and g and a), (e and g and b), (e and g and c), (eand g and d), (e and h and f), (e and h and g), (e and h and i), (e andh and j), (e and h and a), (e and h and b), (e and h and c), (e and hand d), (e and i and f), (e and i and g), (e and i and h), (e and i andj), (e and i and a), (e and i and b), (e and i and c), (e and i and d),(e and j and f), (e and j and g), (e and j and h), (e and j and i), (eand j and a), (e and j and b), (e and j and c), (e and j and d), (f anda and e), (f and a and g), (f and a and h), (f and a and i), (f and aand j), (f and a and b), (f and a and c), (f and a and d), (f and b ande), (f and b and g), (f and b and h), (f and b and i), (f and b and j),(f and b and a), (f and b and c), (f and b and d), (f and c and e), (fand c and g), (f and c and h), (f and c and i), (f and c and j), (f andc and a), (f and c and b), (f and c and d), (f and d and e), (f and dand g), (f and d and h), (f and d and i), (f and d and j), (f and d anda), (f and d and b), (f and d and c), (f and e and d), (f and e and g),(f and e and h), (f and e and i), (f and e and j), (f and e and a), (fand e and b), (f and e and c), (f and g and d), (f and g and e), (f andg and h), (f and g and i), (f and g and j), (f and g and a), (f and gand b), (f and g and c), (f and h and d), (f and h and e), (f and h andg), (f and h and i), (f and h and j), (f and h and a), (f and h and b),(f and h and c), (f and i and d), (f and i and e), (f and i and g), (fand i and h), (f and i and j), (f and i and a), (f and i and b), (f andi and c), (f and j and d), (f and j and e), (f and j and g), (f and jand h), (f and j and i), (f and j and a), (f and j and b), (f and j andc), (g and a and d), (g and a and e), (g and a and f), (g and a and h),(g and a and i), (g and a and j), (g and a and b), (g and a and c), (gand b and d), (g and b and e), (g and b and f), (g and b and h), (g andb and i), (g and b and j), (g and b and a), (g and b and c), (g and cand d), (g and c and e), (g and c and f), (g and c and h), (g and c andi), (g and c and j), (g and c and a), (g and c and b), (g and d and c),(g and d and e), (g and d and f), (g and d and h), (g and d and i), (gand d and j), (g and d and a), (g and d and b), (g and e and c), (g ande and d), (g and e and f), (g and e and h), (g and e and i), (g and eand j), (g and e and a), (g and e and b), (g and f and c), (g and f andd), (g and f and e), (g and f and h), (g and f and i), (g and f and j),(g and f and a), (g and f and b), (g and h and c), (g and h and d), (gand h and e), (g and h and f), (g and h and i), (g and h and j), (g andh and a), (g and h and b), (g and i and c), (g and i and d), (g and iand e), (g and i and f), (g and i and h), (g and i and j), (g and i anda), (g and i and b), (g and j and c), (g and j and d), (g and j and e),(g and j and f), (g and j and h), (g and j and i), (g and j and a), (gand j and b), (h and a and c), (h and a and d), (h and a and e), (h anda and f), (h and a and g), (h and a and i), (h and a and j), (h and aand b), (h and b and c), (h and b and d), (h and b and e), (h and b andf), (h and b and g), (h and b and i), (h and b and j), (h and b and a),(h and c and b), (h and c and d), (h and c and e), (h and c and f), (hand c and g), (h and c and i), (h and c and j), (h and c and a), (h andd and b), (h and d and c), (h and d and e), (h and d and f), (h and dand g), (h and d and i), (h and d and j), (h and d and a), (h and e andb), (h and e and c), (h and e and d), (h and e and f), (h and e and g),(h and e and i), (h and e and j), (h and e and a), (h and f and b), (hand f and c), (h and f and d), (h and f and e), (h and f and g), (h andf and i), (h and f and j), (h and f and a), (h and g and b), (h and gand c), (h and g and d), (h and g and e), (h and g and f), (h and g andi), (h and g and j), (h and g and a), (h and i and b), (h and i and c),(h and i and d), (h and i and e), (h and i and f), (h and i and g), (hand i and j), (h and i and a), (h and j and b), (h and j and c), (h andj and d), (h and j and e), (h and j and f), (h and j and g), (h and jand i), (h and j and a), (i and a and b), (i and a and c), (i and a andd), (i and a and e), (i and a and f), (i and a and g), (i and a and h),(i and a and j), (i and b and a), (i and b and c), (i and b and d), (iand b and e), (i and b and f), (i and b and g), (i and b and h), (i andb and j), (i and c and a), (i and c and b), (i and c and d), (i and cand e), (i and c and f), (i and c and g), (i and c and h), (i and c andj), (i and d and a), (i and d and b), (i and d and c), (i and d and e),(i and d and f), (i and d and g), (i and d and h), (i and d and j), (iand e and a), (i and e and b), (i and e and c), (i and e and d), (i ande and f), (i and e and g), (i and e and h), (i and e and j), (i and fand a), (i and f and b), (i and f and c), (i and f and d), (i and f ande), (i and f and g), (i and f and h), (i and f and j), (i and g and a),(i and g and b), (i and g and c), (i and g and d), (i and g and e), (iand g and f), (i and g and h), (i and g and j), (i and h and a), (i andh and b), (i and h and c), (i and h and d), (i and h and e), (i and hand f), (i and h and g), (i and h and j), (i and j and a), (i and j andb), (i and j and c), (i and j and d), (i and j and e), (i and j and f),(i and j and g), (i and j and h), (j and a and i), (j and a and b), (jand a and c), (j and a and d), (j and a and e), (j and a and f), (j anda and g), (j and a and h), (j and b and i), (j and b and a), (j and band c), (j and b and d), (j and b and e), (j and b and f), (j and b andg), (j and b and h), (j and c and i), (j and c and a), (j and c and b),(j and c and d), (j and c and e), (j and c and f), (j and c and g), (jand c and h), (j and d and i), (j and d and a), (j and d and b), (j andd and c), (j and d and e), (j and d and f), (j and d and g), (j and dand h), (j and e and i), (j and e and a), (j and e and b), (j and e andc), (j and e and d), (j and e and f), (j and e and g), (j and e and h),(j and f and i), (j and f and a), (j and f and b), (j and f and c), (jand f and d), (j and f and e), (j and f and g), (j and f and h), (j andg and i), (j and g and a), (j and g and b), (j and g and c), (j and gand d), (j and g and e), (j and g and f), (j and g and h), (j and h andi), (j and h and a), (j and h and b), (j and h and c), (j and h and d),(j and h and e), (j and h and f), (j and h and g), (j and i and h), (jand i and a), (j and i and b), (j and i and c), (j and i and d), (j andi and e), (j and i and f), and (j and i and g).

2.3. Germlines

The ABPs provided herein may comprise any suitable V_(H) and V_(L)germline sequences.

In some embodiments, the V_(H) region of an ABP provided herein is fromthe VH4 germline. In some embodiments, the V_(H) region of an ABPprovided herein is from the VH1 germline.

In some embodiments, the V_(H) region of an ABP provided herein is fromthe VH4-39 germline. In some embodiments, the V_(H) region of an ABPprovided herein is from the VH4-31 germline. In some embodiments, theV_(H) region of an ABP provided herein is from the VH1-46 germline.

In some embodiments, the V_(L) region of an ABP provided herein is fromthe VK3 germline.

In some embodiments, the V_(L) region of an ABP provided herein is fromthe VK3-11 germline. In some embodiments, the V_(L) region of an ABPprovided herein is from the VK3-20 germline. In some embodiments, theV_(L) region of an ABP provided herein is from the VK3-15 germline.

2.4. Monospecific and Multispecific TIGIT Antigen-Binding Proteins

In some embodiments, the ABPs provided herein are monospecific ABPs.

In some embodiments, the ABPs provided herein are multispecific ABPs.

In some embodiments, a multispecific ABP provided herein binds more thanone antigen. In some embodiments, a multispecific antibody binds 2antigens. In some embodiments, a multispecific antibody binds 3antigens. In some embodiments, a multispecific antibody binds 4antigens. In some embodiments, a multispecific antibody binds 5antigens.

In some embodiments, a multispecific antibody provided herein binds morethan one epitope on a TIGIT antigen. In some embodiments, amultispecific antibody binds 2 epitopes on a TIGIT antigen. In someembodiments, a multispecific antibody binds 3 epitopes on a TIGITantigen.

Many multispecific ABP constructs are known in the art, and the ABPsprovided herein may be provided in the form of any suitablemultispecific suitable construct.

In some embodiments, the multispecific ABP comprises an immunoglobulincomprising at least two different heavy chain variable regions eachpaired with a common light chain variable region (i.e., a “common lightchain antibody”). The common light chain variable region forms adistinct antigen-binding domain with each of the two different heavychain variable regions. See Merchant et al., Nature Biotechnol., 1998,16:677-681, incorporated by reference in its entirety.

In some embodiments, the multispecific ABP comprises an immunoglobulincomprising an antibody or fragment thereof attached to one or more ofthe N- or C-termini of the heavy or light chains of such immunoglobulin.See Coloma and Morrison, Nature Biotechnol., 1997, 15:159-163,incorporated by reference in its entirety. In some aspects, such ABPcomprises a tetravalent bispecific antibody.

In some embodiments, the multispecific ABP comprises a hybridimmunoglobulin comprising at least two different heavy chain variableregions and at least two different light chain variable regions. SeeMilstein and Cuello, Nature, 1983, 305:537-540; and Staerz and Bevan,Proc. Natl. Acad. Sci. USA, 1986, 83:1453-1457; each of which isincorporated by reference in its entirety.

In some embodiments, the multispecific ABP comprises immunoglobulinchains with alterations to reduce the formation of side products that donot have multispecificity. In some aspects, the ABPs comprise one ormore “knobs-into-holes” modifications as described in U.S. Pat. No.5,731,168, incorporated by reference in its entirety.

In some embodiments, the multispecific ABP comprises immunoglobulinchains with one or more electrostatic modifications to promote theassembly of Fc hetero-multimers. See WO 2009/089004, incorporated byreference in its entirety.

In some embodiments, the multispecific ABP comprises a bispecific singlechain molecule. See Traunecker et al., EMBO J., 1991, 10:3655-3659; andGruber et al., J. Immunol., 1994, 152:5368-5374; each of which isincorporated by reference in its entirety.

In some embodiments, the multispecific ABP comprises a heavy chainvariable domain and a light chain variable domain connected by apolypeptide linker, where the length of the linker is selected topromote assembly of multispecific ABPs with the desiredmultispecificity. For example, monospecific scFvs generally form when aheavy chain variable domain and light chain variable domain areconnected by a polypeptide linker of more than 12 amino acid residues.See U.S. Pat. Nos. 4,946,778 and 5,132,405, each of which isincorporated by reference in its entirety. In some embodiments,reduction of the polypeptide linker length to less than 12 amino acidresidues prevents pairing of heavy and light chain variable domains onthe same polypeptide chain, thereby allowing pairing of heavy and lightchain variable domains from one chain with the complementary domains onanother chain. The resulting ABPs therefore have multispecificity, withthe specificity of each binding site contributed by more than onepolypeptide chain. Polypeptide chains comprising heavy and light chainvariable domains that are joined by linkers between 3 and 12 amino acidresidues form predominantly dimers (termed diabodies). With linkersbetween 0 and 2 amino acid residues, trimers (termed triabodies) andtetramers (termed tetrabodies) are favored. However, the exact type ofoligomerization appears to depend on the amino acid residue compositionand the order of the variable domain in each polypeptide chain (e.g.,V_(H)-linker-V_(L) vs. V_(L)-linker-V_(H)), in addition to the linkerlength. A skilled person can select the appropriate linker length basedon the desired multispecificity.

In some embodiments, the multispecific ABP comprises a diabody. SeeHollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90:6444-6448,incorporated by reference in its entirety. In some embodiments, themultispecific ABP comprises a triabody. See Todorovska et al., J.Immunol. Methods, 2001, 248:47-66, incorporated by reference in itsentirety. In some embodiments, the multispecific ABP comprises atetrabody. See id., incorporated by reference in its entirety.

In some embodiments, the multispecific ABP comprises a trispecificF(ab′)3 derivative. See Tutt et al. J. Immunol., 1991, 147:60-69,incorporated by reference in its entirety.

In some embodiments, the multispecific ABP comprises a cross-linkedantibody. See U.S. Pat. No. 4,676,980; Brennan et al., Science, 1985,229:81-83; Staerz, et al. Nature, 1985, 314:628-631; and EP 0453082;each of which is incorporated by reference in its entirety.

In some embodiments, the multispecific ABP comprises antigen-bindingdomains assembled by leucine zippers. See Kostelny et al., J. Immunol.,1992, 148:1547-1553, incorporated by reference in its entirety.

In some embodiments, the multispecific ABP comprises complementaryprotein domains. In some aspects, the complementary protein domainscomprise an anchoring domain (AD) and a dimerization and docking domain(DDD). In some embodiments, the AD and DDD bind to each other andthereby enable assembly of multispecific ABP structures via the “dockand lock” (DNL) approach. ABPs of many specificities may be assembled,including bispecific ABPs, trispecific ABPs, tetraspecific ABPs,quintspecific ABPs, and hexaspecific ABPs. Multispecific ABPs comprisingcomplementary protein domains are described, for example, in U.S. Pat.Nos. 7,521,056; 7,550,143; 7,534,866; and 7,527,787; each of which isincorporated by reference in its entirety.

In some embodiments, the multispecific ABP comprises a dual action Fab(DAF) antibody as described in U.S. Pat. Pub. No. 2008/0069820,incorporated by reference in its entirety.

In some embodiments, the multispecific ABP comprises an antibody formedby reduction of two parental molecules followed by mixing of the twoparental molecules and reoxidation to assembly a hybrid structure. SeeCarlring et al., PLoS One, 2011, 6:e22533, incorporated by reference inits entirety.

In some embodiments, the multispecific ABP comprises a DVD-Ig™. ADVD-Ig™ is a dual variable domain immunoglobulin that can bind to two ormore antigens. DVD-Igs™ are described in U.S. Pat. No. 7,612,181,incorporated by reference in its entirety.

In some embodiments, the multispecific ABP comprises a DART™. DARTs™ aredescribed in Moore et al., Blood, 2011, 117:454-451, incorporated byreference in its entirety.

In some embodiments, the multispecific ABP comprises a DuoBody®.DuoBodies® are described in Labrijn et al., Proc. Natl. Acad. Sci. USA,2013, 110:5145-5150; Gramer et al., mAbs, 2013, 5:962-972; and Labrijnet al., Nature Protocols, 2014, 9:2450-2463; each of which isincorporated by reference in its entirety.

In some embodiments, the multispecific ABP comprises an antibodyfragment attached to another antibody or fragment. The attachment can becovalent or non-covalent. When the attachment is covalent, it may be inthe form of a fusion protein or via a chemical linker Illustrativeexamples of multispecific ABPs comprising antibody fragments attached toother antibodies include tetravalent bispecific antibodies, where anscFv is fused to the C-terminus of the C_(H3) from an IgG. See Colomaand Morrison, Nature Biotechnol., 1997, 15:159-163. Other examplesinclude antibodies in which a Fab molecule is attached to the constantregion of an immunoglobulin. See Miler et al., J. Immunol., 2003,170:4854-4861, incorporated by reference in its entirety. Any suitablefragment may be used, including any of the fragments described herein orknown in the art.

In some embodiments, the multispecific ABP comprises a CovX-Body.CovX-Bodies are described, for example, in Doppalapudi et al., Proc.Natl. Acad. Sci. USA, 2010, 107:22611-22616, incorporated by referencein its entirety.

In some embodiments, the multispecific ABP comprises an Fcab antibody,where one or more antigen-binding domains are introduced into an Fcregion. Fcab antibodies are described in Wozniak-Knopp et al., ProteinEng. Des. Sel., 2010, 23:289-297, incorporated by reference in itsentirety.

In some embodiments, the multispecific ABP comprises an TandAb®antibody. TandAb® antibodies are described in Kipriyanov et al., J. Mol.Biol., 1999, 293:41-56 and Zhukovsky et al., Blood, 2013, 122:5116, eachof which is incorporated by reference in its entirety.

In some embodiments, the multispecific ABP comprises a tandem Fab.Tandem Fabs are described in WO 2015/103072, incorporated by referencein its entirety.

In some embodiments, the multispecific ABP comprises a Zybody™.Zybodies™ are described in LaFleur et al., mAbs, 2013, 5:208-218,incorporated by reference in its entirety.

2.5. TIGIT Antagonism

In some embodiments, the ABPs provided herein antagonize TIGIT uponbinding.

In some embodiments, antagonism of TIGIT by an ABP provided hereinresults in dimerization and/or activation of CD226 (also known asDNAM-1), a co-stimulatory receptor whose dimerization and function isimpaired by direct interaction with TIGIT. See Grogan et al., J.Immunol., 2014, 192 (1 Supplement) 2013.15, incorporated by reference inits entirety. FIG. 2 provides an illustration of the CD226-TIGIT pathwayin comparison to the CD28/CTLA4 pathway, which has similarcostimulation/coinhibition biology.

In some embodiments, antagonism of TIGIT by an ABP provided hereinincreases the amount of CD226 and CD155 that interact in comparison tothe amount that interact in the absence of the ABP.

In some embodiments, antagonism of TIGIT by an ABP provided hereinresults in activation of an effector T cell. In some aspects, theeffector T cell is a CD8+ T cell. In some aspects, the effector T cellis a CD4+ T cell.

In some embodiments, antagonism of TIGIT by an ABP provided hereinresults in activation of an NK cell. In some embodiments, antagonism ofTIGIT by an ABP provided herein results in activation of an NKT cell.

In some embodiments, antagonism of TIGIT by an ABP provided hereinresults in a reduction of the inhibitory activity of a regulatory T celltoward an effector T cell.

In some embodiments, antagonism of TIGIT by an ABP provided hereinresults in increased secretion of IL-2, IL-6, GM-CSF, TNF, LT-α, and/orIFN-γ by a target cell.

In some embodiments, antagonism of TIGIT by an ABP provided hereinincreases the proliferation, survival, and/or function of an effector Tcell. In some aspects the effector T cell is a CD4+ effector T cell. Insome aspects, the effector T cell is a CD8+ effector T cell.

In some embodiments, antagonism of TIGIT by an ABP provided hereinabrogates suppression of an effector T cell by a regulatory T cell. Insome aspects, the regulatory T cell is a CD4+CD25+Foxp3+ regulator Tcell. In some aspects, the regulatory T cell is a CD8+CD25+ regulatory Tcell.

In some embodiments, antagonism of TIGIT by an ABP provided hereinresults in an enhancement of an immune response.

In some embodiments, antagonism of TIGIT by an ABP provided hereinresults in the prevention of a tumor. In some embodiments, antagonism ofTIGIT by an ABP provided herein results in the delay of onset of atumor. In some embodiments, antagonism of TIGIT by an ABP providedherein results in a reduction of the size of a tumor. In someembodiments, antagonism of TIGIT by an ABP provided herein results inelimination of a tumor. In some embodiments, antagonism of TIGIT by anABP provided herein results in a reduction in the number of metastases.

In some embodiments, antagonism of TIGIT by an ABP provided hereinresults in the prevention of a viral disease. In some embodiments,antagonism of TIGIT by an ABP provided herein results in the delay ofonset of a viral disease. In some embodiments, antagonism of TIGIT by anABP provided herein results in a reduction of the viral load in asubject. In some embodiments, antagonism of TIGIT by an ABP providedherein results in the elimination of a viral infection.

2.6. Affinity and Kinetics of Antigen-Binding Proteins for TIGIT;Potency

In some embodiments, the affinity of an ABP provided herein for TIGIT asindicated by K_(D), is less than about 10⁻⁵ M, less than about 10⁻⁶ M,less than about 10⁻⁷ M, less than about 10⁻⁸ M, less than about 10⁻⁹ M,less than about 10⁻¹⁰ less than about 10⁻¹¹ M, or less than about 10⁻¹²M. In some M, embodiments, the affinity of the ABP is between about 10⁻⁷M and 10⁻¹² M. In some embodiments, the affinity of the ABP is betweenabout 10⁻⁷ M and 10⁻¹¹ M. In some embodiments, the affinity of the ABPis between about 10⁻⁷ M and 10⁻¹⁰ M. In some embodiments, the affinityof the ABP is between about 10⁻⁷ M and 10⁻⁹ M. In some embodiments, theaffinity of the ABP is between about 10⁻⁷ M and 10⁻⁸ M. In someembodiments, the affinity of the ABP is between about 10⁻⁸ M and 10⁻¹²M. In some embodiments, the affinity of the ABP is between about 10⁻⁸ Mand 10⁻¹¹ M. In some embodiments, the affinity of the ABP is betweenabout 10⁻⁹ M and 10⁻¹¹ M. In some embodiments, the affinity of the ABPis between about 10⁻¹⁰ M and 10⁻¹¹ M.

In some embodiments, the affinity of an ABP provided herein for hTIGITas indicated by K_(D) measured by ForteBio, as set forth in Example 4 isselected from about 5.24×10⁻¹⁰ M, about 4.57×10⁻¹⁰ M, about 3.32×10⁻¹⁰about 2.46×10⁻¹⁰ M, about 1.96×10⁻¹⁰ M, about 3.11×10⁻⁹ M, about M,2.54×10 M, about 3.13×10 M, about 2.83×10⁻⁹ M, about 1.71×10⁻⁹ M, about2.47×10⁻⁹ M, about 2.35×10 M, about 1.44×10 M, about 1.23×10⁻⁹ M, about5.26×10⁻¹⁰ M, about 3.78×10⁻¹° M, about 4.29×10⁻¹⁰ M, or about4.48×10⁻¹⁰ M. In some embodiments, such affinity ranges from about3.13×10⁻⁹ M to about 1.96×10⁻¹⁰ M. In some embodiments, such K_(D) isabout 3.13×10⁻⁹ M or less.

In some embodiments, the affinity of an ABP provided herein for cTIGITas indicated by K_(D) measured by ForteBio, as set forth in Example 4 isselected from about 2.64×10⁻⁹ M, about 6.55×10⁻⁹ M, about 8.14×10 M,about 6.57×10⁻⁹ M, about 7.94×10⁻⁸ M, about 7.04×10⁻⁸ M, about 1.10×10⁻⁷M, about 7.20×10⁻⁸ M, about 1.57×10⁻⁹ M, about 8.02×10⁻¹⁰ M, about3.67×10⁻¹⁰ M, about 8.98×10⁻¹⁰ M, about 1.75×10⁻⁸ M, or about 2.58×10⁻⁸M, about 9.35×10⁻⁹ M. In some embodiments, such affinity ranges fromabout 1.10×10⁻⁷ M to about 3.69×10⁻¹⁰ M. In some embodiments, such K_(D)is about 1.10×10⁻⁷M or less.

In some embodiments, the affinity of an ABP provided herein for hTIGITas indicated by K_(D) measured by solution equilibrium methods(MSD-SET), as set forth in Example 4 is selected from about 5.40×10⁻¹¹M, about 1.10×10⁻¹⁰ M, about 1.50×10⁻¹⁰ M, about 5.60×10⁻¹¹ M, about4.00×10⁻¹⁰ M, about 3.80×10⁻¹⁰ M, about 2.10×10⁻¹⁰ M, about 7.00×10⁻¹¹M, about 4.10×10⁻¹¹ M, about 2.50×10⁻¹¹ M, about 3.00×10⁻¹¹ M, about8.00×10⁻¹¹ M, about 8.10×10⁻¹² M, about 5.00×10⁻¹² M, or about4.90×10⁻¹² M. In some embodiments, such affinity ranges from about4.00×10⁻¹⁰ M to about 4.90×10⁻¹² M. In some embodiments, such K_(D) isabout 4.00×10⁻¹⁰ M or less.

In some embodiments, the affinity of an ABP provided herein for cTIGITas indicated by K_(D) measured by MSD-SET, as set forth in Example 4 isselected from about 3.20×10⁻¹⁰ M, about 2.30×10⁻¹⁰ M, about 3.50×10⁻¹¹M, about 1.50×10⁻¹¹ M, or about 4.60×10⁻¹¹ M. In some embodiments, suchaffinity ranges from about 3.20×10⁻¹⁰ M to about 1.50×10⁻¹¹ M. In someembodiments, such K_(D) is about 3.20×10⁻¹⁰ M or less.

In some embodiments, the affinity of an ABP provided herein for hTIGITas indicated by K_(D) measured by ForteBio, as set forth in Example 6 isselected from about 7.1×10⁻¹⁰ M, about 8.1×10⁻¹¹M, about 1.9×10⁻¹⁰ M,about 5.6×10⁻¹⁰ M, about 2.4×10⁻¹⁰ M, about 2.8×10⁻¹⁰ M, about 1.6×10⁻¹⁰M, about 5.8×10⁻¹⁰ M, about 1.1×10⁻⁹ M, about 8.1×10⁻¹⁰ M, about4.6×10⁻¹⁰ M, or about 3.6×10⁻¹⁰ M. In some embodiments, such affinityranges from about 1.1×10⁻⁹ M to about 8.1×10⁻¹¹ M. In some embodiments,such K_(D) is about 1.1×10⁻⁹ M or less.

In some embodiments, the affinity of an ABP provided herein for hTIGITas indicated by K_(D) measured by ForteBio, as set forth in Example 6,is about 2.4×10⁻¹⁰ M. In some embodiments, the affinity of an ABPprovided herein for cTIGIT as indicated by K_(D) measured by ForteBio,as set forth in Example 6, is about 6.2×10⁻⁹ M. In some embodiments,such K_(D) is about 6.2×10⁻⁹ M or less.

In some embodiments, the affinity of an ABP provided herein for hTIGITexpressed on the surface of a Jurkat cell, as indicated by K_(D),described in Example 6 is about 5.1×10⁻¹⁰ M. In some embodiments, suchK_(D) is about 5.1×10⁻¹⁰ M or less.

In some embodiments, the affinity of an ABP provided herein for cTIGITexpressed on the surface of a Jurkat cell, as indicated by K_(D),described in Example 6 is about 4.0×10⁻¹⁰ M. In some embodiments, suchK_(D) is about 4.0×10⁻¹⁰ M or less.

In some embodiments, the affinity of an ABP provided herein for mTIGIT(SEQ ID NO: 3) expressed on the surface of a Jurkat cell, as indicatedby K_(D), described in Example 6 is about 9.8×10⁻⁹ M. In someembodiments, such K_(D) is about 9.8×10⁻⁹ M or less. In someembodiments, such K_(D) is about 9.8×10⁻⁹ M or greater.

In some embodiments, the affinity of an ABP provided herein for hTIGITexpressed on the surface of a human CD8+ T cell, as indicated by K_(D),described in Example 6 is about 1.3×10⁻⁹ M. In some embodiments, suchK_(D) is about 1.3×10⁻⁹ M or less.

In some embodiments, the affinity of an ABP provided herein for cTIGITexpressed on the surface of a cynomolgus monkey CD8+ T cell, asindicated by K_(D), described in Example 6 is about 2.8×10⁻⁹ M. In someembodiments, such K_(D) is about 2.8×10⁻⁹ M or less.

In some embodiments, the affinity of an ABP provided herein for mTIGITexpressed on the surface of a murine T regulatory cell (i.e., mTIGIT asit naturally occurs on such cells, whether or not such mTIGIT is of SEQID NOs: 3 or 138, but inclusive of such SEQ ID NOs), as indicated byK_(D), described in Example 6 is about 2.5×10⁻⁸ M. In some embodiments,such K_(D) is about 2.5×10⁻⁸ M or less.

In some embodiments, the ABPs provided herein specifically bind tohTIGIT (SEQ ID NO:1) with a K_(D) of X and to cTIGIT (SEQ ID NO:2) ormTIGIT (SEQ ID NO:3 or 138) with a K_(D) of ≤10×. In some embodiments,the ABPs provided herein specifically bind to hTIGIT (SEQ ID NO:1) witha K_(D) of X and to cTIGIT (SEQ ID NO:2) or mTIGIT (SEQ ID NO:3 or 138)with a K_(D) of ≤5×. In some embodiments, the ABPs provided hereinspecifically bind to hTIGIT (SEQ ID NO:1) with a K_(D) of X and tocTIGIT (SEQ ID NO:2) or mTIGIT (SEQ ID NO:3 or 138) with a K_(D) of ≤2×.In some aspects, X is any K_(D) described in this disclosure. In someaspects, X is 0.01 nM, 0.1 nM, 1 nM, 10 nM, 20 nM, 50 nM, or 100 nM.

In some embodiments, the ratio of K_(D(hTIGIT)):K_(D(cTIGIT)) for an ABPprovided herein, as measured by ForteBio as set forth in Example 4, isselected from about 1.98×10⁻¹, about 2.61×10⁻¹, about 3.03×10⁻¹, about3.58×10⁻¹, about 6.62×10⁻³, about 1.98×10⁻¹, about 5.37×10⁻³, about3.90×10⁻³, about 6.22×10⁻³, about 2.91×10⁻¹, about 4.14×10⁻¹, about6.67×10⁻¹, about 2.18×10⁻¹, about 1.78×10⁻¹, about 1.21×10⁻¹, or about3.03×10⁻¹. In some embodiments, such ratio ranges from about 3.90×10⁻³to about 6.67×10⁻¹.

In some embodiments, the ratio of K_(D(hTIGIT)):K_(D(cTIGIT)) for an ABPprovided herein, as measured by ForteBio as set forth in Example, isabout 3.87×10⁻².

In some embodiments, the ratio of K_(D(hTIGIT)):K_(D(cTIGIT)) for an ABPprovided herein, as measured by MSD-SET as set forth in Example 4, isselected from about 3.33×10⁻¹, about 2.31×10⁻¹, about 1.09×10⁻¹, about1.07×10⁻¹, or about 1.69×10⁻¹. In some embodiments, such ratio rangesfrom about 1.07×10⁻¹ M to about 3.33×10⁻¹ M.

In some embodiments an ABP provided herein has a k_(a) of at least about10⁴ M⁻¹×sec⁻¹. In some embodiments the ABP has a k_(a) of at least about10⁵ M⁻¹×sec⁻¹. In some embodiments the ABP has a k_(a) of at least about10⁶ M⁻¹×sec⁻¹. In some embodiments the ABP has a k_(a) of between about10⁴ M⁻¹×sec⁻¹ and about 10⁵ M⁻¹×sec⁻¹. In some embodiments the ABP has ak_(a) of between about 10⁵ M⁻¹×sec⁻¹ and about 10⁶ M⁻¹×sec⁻¹. In someembodiments, such k_(a) is at least about 10⁵ M⁻¹×sec⁻¹.

In some embodiments, an ABP provided herein has a k_(a) for hTIGIT, asmeasured by ForteBio as set forth in Example 6, selected from about3.2×10⁵ M⁻¹×sec⁻¹, about 7.0×10⁵ M⁻¹×sec⁻¹, about 7.7×10⁵ M⁻¹×sec⁻¹,about 1.6×10⁶ M⁻¹×sec⁻¹, about 2.0×10⁶ M⁻¹×sec⁻¹, about 1.3×10⁶M⁻¹×sec⁻¹, about 1.5×10⁶ M⁻¹×sec⁻¹, about 1.1×10⁶ M⁻¹×sec⁻¹, about4.5×10⁵ M⁻¹×sec⁻¹, about 7.5×10⁵ M⁻¹×sec⁻¹, about 8.9×10⁵ M⁻¹×sec⁻¹, orabout 1.4×10⁶ M⁻¹×sec⁻¹. In some embodiments, such k_(a) ranges fromabout 3.2×10⁵ M⁻¹×sec⁻¹ to about 2.0×10⁶ M⁻¹×sec. In some embodiments,such k_(d) is about 2.0×10⁶ M or less.

In some embodiments, an ABP provided herein has a k_(a) for hTIGIT, asmeasured by ForteBio as set forth in Example 6, of about 2.0×10⁶M⁻¹×sec⁻¹. In some embodiments, such k_(a) is at least about 2.0×10⁶M⁻¹×sec⁻¹.

In some embodiments, an ABP provided herein has a k_(a) for cTIGIT, asmeasured by ForteBio as set forth in Example 6, of about 7.9×10⁵M⁻¹×sec⁻¹. In some embodiments, such k_(a) is at least about 7.9×10⁵M⁻¹×sec⁻¹.

In some embodiments an ABP provided herein has a k_(d) of about 10⁻⁵sec⁻¹ or less. In some embodiments the ABP has a k_(d) of about 10⁻⁴sec⁻¹ or less. In some embodiments the ABP has a k_(d) of about 10⁻³sec⁻¹ or less. In some embodiments the ABP has a k_(d) of between about10⁻² sec⁻¹ and about 10⁻⁵ sec⁻¹. In some embodiments the ABP has a k_(d)of between about 10⁻² sec⁻¹ and about 10⁻⁴ sec⁻¹. In some embodimentsthe ABP has a k_(d) of between about 10⁻³ sec⁻¹ and about 10⁻⁵ sec⁻¹.

In some embodiments, an ABP provided herein has a k_(d), for hTIGIT, asmeasured by ForteBio as set forth in Example 6, selected from about2.3×10⁻⁴ sec⁻¹, about 6.3×10⁻⁵ sec⁻¹, about 1.4×10⁻⁴ sec⁻¹, about8.5×10⁻⁴ sec⁻¹, about 3.8×10⁻⁴ sec⁻¹, about 3.5×10⁻⁴ sec⁻¹, about2.4×10⁻⁴ sec⁻¹, about 6.6×10⁻⁴ sec⁻¹, about 5.9×10⁻⁴ sec⁻¹, or about5.0×10⁻⁴ sec⁻¹. In some embodiments, such k_(d) ranges from about6.3×10⁻⁵ sec⁻¹ to about 8.5×10⁻⁴ sec⁻¹. In some embodiments, such k_(d)is less than about 8.5×10⁻⁴ sec⁻¹.

In some embodiments, an ABP provided herein has a k_(d) for hTIGIT, asmeasured by ForteBio as set forth in Example 6, of about 3.8×10⁻⁴ sec⁻¹.In some embodiments, such k_(d) is less than about 3.8×10⁻⁴ sec⁻¹.

In some embodiments, an ABP provided herein has a k_(d) for cTIGIT, asmeasured by ForteBio as set forth in Example 6, of about 4.6×10⁻³ sec⁻¹.In some embodiments, such k_(d) is less than about 4.6×10⁻³ sec⁻¹.

In some embodiments, an ABP provided herein has a k_(a) for hTIGIT ofabout 3.2×10⁵ M⁻¹×sec⁻¹, a k_(d) for hTIGIT of about 2.3×10⁻⁴ sec⁻¹, anda K_(D) for hTIGIT of about 7.1×10⁻¹⁰ M. In some embodiments, an ABPprovided herein has a k_(a) for hTIGIT of about 7.0×10⁵ M⁻¹×sec⁻¹, ak_(d) for hTIGIT of about 6.3×10⁻⁵ sec⁻¹, and a K_(D) for hTIGIT ofabout 8.1×10⁻¹¹ M. In some embodiments, an ABP provided herein has ak_(a) for hTIGIT of about 7.7×10⁵ M⁻¹×sec⁻¹, a k_(d) for hTIGIT of about1.4×10⁻⁴ sec⁻¹, and a K_(D) for hTIGIT of about 1.9×10⁻¹⁰ M. In someembodiments, an ABP provided herein has a k_(a) for hTIGIT of about1.6×10⁶ M⁻¹×sec⁻¹, a k_(d) for hTIGIT of about 8.5×10⁻⁴ sec⁻¹, and aK_(D) for hTIGIT of about 5.6×10⁻¹⁰ M. In some embodiments, an ABPprovided herein has a k_(a) for hTIGIT of about 2.0×10⁶ M⁻¹×sec⁻¹, ak_(d) for hTIGIT of about 3.8×10⁻⁴ sec⁻¹, and a K_(D) for hTIGIT ofabout 2.4×10⁻¹⁰ M. In some embodiments, an ABP provided herein has ak_(a) for hTIGIT of about 1.3×10⁶ M⁻¹×sec⁻¹, a k_(d) for hTIGIT of about3.5×10⁻⁴ sec⁻¹, and a K_(D) for hTIGIT of about 2.8×10⁻¹⁰ M. In someembodiments, an ABP provided herein has a k_(a) for hTIGIT of about1.5×10⁶ M⁻¹×sec⁻¹, a k_(d) for hTIGIT of about 2.4×10⁻⁴ sec⁻¹, and aK_(D) for hTIGIT of about 1.6×10⁻¹⁰ M. In some embodiments, an ABPprovided herein has a k_(a) for hTIGIT of about 1.1×10⁶ M⁻¹×sec⁻¹, ak_(d) for hTIGIT of about 6.6×10⁻⁴ sec⁻¹, and a K_(D) for hTIGIT ofabout 5.8×10⁻¹⁰ M. In some embodiments, an ABP provided herein has ak_(a) for hTIGIT of about 4.5×10⁵ M⁻¹×sec⁻¹, a k_(d) for hTIGIT of about3.5×10⁻⁴ sec⁻¹, and a K_(D) for hTIGIT of about 1.1×10⁻⁹ M. In someembodiments, an ABP provided herein has a k_(a) for hTIGIT of about7.5×10⁵ M⁻¹×sec⁻¹, a k_(d) for hTIGIT of about 5.9×10⁻⁴ sec⁻¹, and aK_(D) for hTIGIT of about 8.1×10⁻¹⁰ M. In some embodiments, an ABPprovided herein has a k_(a) for hTIGIT of about 8.9×10⁵ M⁻¹×sec⁻¹, ak_(d) for hTIGIT of about 3.8×10⁻⁴ sec⁻¹, and a K_(D) for hTIGIT ofabout 4.6×10⁻¹⁰ M. In some embodiments, an ABP provided herein has ak_(a) for hTIGIT of about 1.4×10⁶ M⁻¹×sec⁻¹, a k_(d) for hTIGIT of about5.0×10⁻⁴ sec⁻¹, and a K_(D) for hTIGIT of about 3.6×10⁻¹⁰ M. In someembodiments, such k_(a), k_(d) and K_(D) are determined according to themethods provided in Example 6.

In some embodiments, an ABP provided herein has a k_(a) for hTIGIT ofabout 2.0×10⁶ M⁻¹×sec⁻¹, a k_(d) for hTIGIT of about 3.8×10⁻⁴ sec⁻¹, aK_(D) for hTIGIT of about 2.4×10⁻¹⁰ M, a k_(a) for cTIGIT of about7.9×10⁵ M⁻¹×sec⁻¹, a k_(d) for cTIGIT of about 4.6×10⁻³ sec⁻¹, a K_(D)for cTIGIT of about 6.2×10⁻⁹ M, and a K_(D) for mTIGIT (SEQ ID NO: 3) ofgreater than about 7.0×10⁻⁷ M. In some embodiments, such k_(a), k_(d)and K_(D) are determined according to the methods provided in Example 6.

In some embodiments, K_(D), k_(a), and k_(d) are determined usingsurface plasmon resonance (SPR). In some aspects, the SPR analysisutilizes a BIACORE® instrument. In some aspects, the antigen isimmobilized on a carboxymethylated dextran biosensor chip (CM4 or CM5)and contacted with an ABP provided herein. Association and dissociationrate constants may be calculated using the BIAevaluation® software and aone-to-one Langmuir binding model. In some aspects, the assay isperformed at 25° C. In some aspects, the assay is performed at 37° C.

In some embodiments, K_(D), k_(a), and k_(d) are determined usingbiolayer interferometry (BLI). Any suitable BLI method may be used. Insome aspects, the BLI analysis utilizes a FORTEBIO® instrument. In someaspects, an anti-human IgG Fc capture (AHC) biosensor is used to captureABPs onto the surface of a sensor. Subsequently, association of the ABPand antigen is monitored by contacting the immobilized ABP withdifferent concentrations of TIGIT. Dissociation of the antigen and ABPis then measured in a buffer without TIGIT. Association and dissociationrate constants are calculated using the kinetic modules of the FORTEBIO®Analysis Software. In some aspects, the assay is performed at 30° C.

In other embodiments, K_(D) may be determined by a radiolabeledantigen-binding assay, as described in Chen et al. J. Mol. Biol., 1999,293:865-881, incorporated by reference in its entirety.

In other embodiments, K_(D) may be determined by using MSD-SET, asdescribed in Example 4.

In some embodiments, an ABP provided herein has an EC₅₀, as measured byIL-2 production in a human TIGIT Jurkat co-culture assay as described inExample 7, of about 0.22 nM, about 0.31 nM, about 0.33 nM, about 0.34nM, about 0.25 nM, about 0.24 nM, about 0.11 nM, about 0.06 nM, about0.14 nM, about 0.16 nM, about 1.40 nM, about 0.71 nM, about 0.21 nM,about 1.11 nM, about 0.13 nM, about 0.20 nM, about 0.68 nM, or about0.61 nM. In some embodiments, such EC₅₀ ranges from about 0.06 nM toabout 1.40 nM. In some embodiments, such EC₅₀ is about 1.40 nM or less.

In some embodiments, an ABP provided herein has an EC₅₀, as measured byIL-2 production in a cynomolgus monkey TIGIT Jurkat co-culture assay asdescribed in Example 7, of about 2.87 nM. In some embodiments, such EC₅₀is about 2.87 nM or less. In some embodiments, the ratio ofEC_(50(cTIGIT)):EC_(50(hTIGIT)) in such assay ranges from about 2.05 toabout 47.8.

In some embodiments, an ABP provided herein has an EC₁₀, as measured byTNF production in PBMCs isolated from a human donor and treated asdescribed in Example 9, in a range of about 5.02 nM to about 18.86 nM.In some embodiments, such EC₁₀ is about 18.86 nM or less.

In some embodiments, an ABP provided herein has an EC₅₀, as measured byTNF production in PBMCs isolated from a human donor and treated asdescribed in Example 9, in a range of about 12.60 nM to about 20.60 nM.In some embodiments, such EC₅₀ is about 20.60 nM or less.

In some embodiments, an ABP provided herein has an EC₉₀, as measured byTNF production in PBMCs isolated from a human donor and treated asdescribed in Example 9, in a range of about 22.49 nM to about 31.59 nM.In some embodiments, such EC₉₀ is about 31.59 nM or less.

In some embodiments, an ABP provided herein has an EC₁₀ in a range ofabout 5.02 nM to about 18.86 nM, an EC₅₀ in a range of about 12.60 nM toabout 20.60 nM, and EC₉₀ in a range of about 22.49 nM to about 31.59 nM,in each cases as measured by TNF production in PBMCs isolated from ahuman donor and treated as described in Example 9.

In some embodiments, an ABP provided herein has an EC₁₀ of about 11.94nM or less, an EC₅₀ of about 16.60 nM or less, and EC₉₀ of about 27.04nM or less, in each cases as measured by TNF production in PBMCsisolated from a human donor and treated as described in Example 9.

In some embodiments, an ABP provided herein has an EC₁₀ of about 18.86nM or less, an EC₅₀ of about 20.06 nM or less, and EC₉₀ of about 31.59nM or less, in each cases as measured by TNF production in PBMCsisolated from a human donor and treated as described in Example 9.

In some embodiments, an ABP provided herein has an EC₁₀ of about 5.02 nMor less, an EC₅₀ of about 12.60 nM or less, and EC₉₀ of about 22.49 nMor less, in each cases as measured by TNF production in PBMCs isolatedfrom a human donor and treated as described in Example 9.

In some embodiments, an ABP provided herein has an EC₁₀, as measured byIFN-γ production in CD4+ T cells isolated from a human donor and treatedas described in Example 9, in a range of about 0.37 nM to about 1.05 nM.In some embodiments, such EC₁₀ is about 1.05 nM or less.

In some embodiments, an ABP provided herein has an EC₅₀, as measured byIFN-γ production in CD4+ T cells isolated from a human donor and treatedas described in Example 9, in a range of about 0.94 nM to about 1.12 nM.In some embodiments, such EC₅₀ is about 1.12 nM or less.

In some embodiments, an ABP provided herein has an EC₉₀, as measured byIFN-γ production in CD4+ T cells isolated from a human donor and treatedas described in Example 9, in a range of about 1.04 nM to about 2.72 nM.In some embodiments, such EC₉₀ is about 2.72 nM or less.

In some embodiments, an ABP provided herein has an EC₁₀ in a range ofabout 0.37 nM to about 1.05 nM, an EC₅₀ in a range of about 0.94 nM toabout 1.12 nM, and EC₉₀ in a range of about 1.04 nM to about 2.72 nM, ineach cases as measured by IFN-γ production in PBMCs isolated from ahuman donor and treated as described in Example 9.

In some embodiments, an ABP provided herein has an EC₁₀ of about 0.37 nMor less, an EC₅₀ of about 1.00 nM or less, and EC₉₀ of about 2.72 nM orless, in each cases as measured by IFN-γ production in PBMCs isolatedfrom a human donor and treated as described in Example 9.

In some embodiments, an ABP provided herein has an EC₁₀ of about 0.85 nMor less, an EC₅₀ of about 0.94 nM or less, and EC₉₀ of about 1.04 nM orless, in each cases as measured by IFN-γ production in PBMCs isolatedfrom a human donor and treated as described in Example 9.

In some embodiments, an ABP provided herein has an EC₁₀ of about 1.05 nMor less, an EC₅₀ of about 1.12 nM or less, and EC₉₀ of about 1.19 nM orless, in each cases as measured by IFN-γ production in PBMCs isolatedfrom a human donor and treated as described in Example 9.

In some embodiments, an ABP provided herein has an EC₁₀ of about 0.75 nMor less, an EC₅₀ of about 1.02 nM or less, and EC₉₀ of about 1.65 nM orless, in each cases as measured by IFN-γ production in PBMCs isolatedfrom a human donor and treated as described in Example 9.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 5.24×10⁻¹⁰ M (as determined by ForteBio), cTIGIT with a K_(D) ofabout 2.64×10⁻⁹ M (as determined by ForteBio), hTIGIT with a K_(D) ofabout 5.40×10⁻¹¹ M (as determined by MSD-SET), and cTIGIT with a K_(D)of about 3.20×10⁻¹⁰ M (as determined by MSD-SET), in each casedetermined according to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 4.57×10⁻¹⁰ M (as determined by ForteBio), cTIGIT with a K_(D) ofabout 1.57×10⁻⁹ M (as determined by ForteBio), hTIGIT with a K_(D) ofabout 2.50×10⁻¹¹ M (as determined by MSD-SET), and cTIGIT with a K_(D)of about 2.30×10⁻¹⁰ M (as determined by MSD-SET), in each casedetermined according to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 3.32×10⁻¹⁰ M (as determined by ForteBio), cTIGIT with a K_(D) ofabout 8.02×10⁻¹⁰ M (as determined by ForteBio), hTIGIT with a K_(D) ofabout 8.10×10⁻¹² M (as determined by MSD-SET), and cTIGIT with a K_(D)of about 3.50×10⁻¹ M (as determined by MSD-SET), in each case determinedaccording to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 2.46×10⁻¹⁰ M (as determined by ForteBio), cTIGIT with a K_(D) ofabout 3.69×10⁻¹⁰ M (as determined by ForteBio), hTIGIT with a K_(D) ofabout 5.00×10⁻¹² M (as determined by MSD-SET), and cTIGIT with a K_(D)of about 1.50×10⁻¹¹ M (as determined by MSD-SET), in each casedetermined according to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 1.96×10⁻¹⁰ M (as determined by ForteBio), cTIGIT with a K_(D) ofabout 8.98×10⁻¹⁰ M (as determined by ForteBio), hTIGIT with a K_(D) ofabout 4.90×10⁻¹² M (as determined by MSD-SET), and cTIGIT with a K_(D)of about 4.60×10⁻¹¹ M (as determined by MSD-SET), in each casedetermined according to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 3.11×10⁻⁹ M (as determined by ForteBio) and cTIGIT with a K_(D) ofabout 1.75×10⁻⁸ M (as determined by ForteBio), in each case determinedaccording to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 2.54×10⁻⁹ M, as determined by ForteBio according to the methodsprovided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 3.13×10⁻⁹ M (as determined by ForteBio) and cTIGIT with a K_(D) ofabout 2.58×10⁻⁸ M (as determined by ForteBio), in each case determinedaccording to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 2.83×10⁻⁹ M (as determined by ForteBio) and cTIGIT with a K_(D) ofabout 9.35×10⁻⁹ M (as determined by ForteBio), in each case determinedaccording to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 1.71×10⁻⁹ M (as determined by ForteBio), cTIGIT with a K_(D) ofabout 6.55×10⁻⁹ M (as determined by ForteBio), and hTIGIT with a K_(D)of about 1.10×10⁻¹⁰ M (as determined by MSD-SET), in each casedetermined according to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 2.47×10⁻⁹ M (as determined by ForteBio), cTIGIT with a K_(D) ofabout 8.14×10⁻⁹ M (as determined by ForteBio), and hTIGIT with a K_(D)of about 1.50×10⁻¹⁰ M (as determined by MSD-SET), in each casedetermined according to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 2.35×10⁻⁹ M (as determined by ForteBio), cTIGIT with a K_(D) ofabout 6.57×10⁻⁹ M (as determined by ForteBio), and hTIGIT with a K_(D)of about 5.60×10⁻¹¹ M (as determined by MSD-SET), in each casedetermined according to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 1.44×10⁻⁹ M (as determined by ForteBio) and hTIGIT with a K_(D) ofabout 4.00×10⁻¹⁰ M (as determined by MSD-SET), in each case determinedaccording to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 1.23×10⁻⁹ M (as determined by ForteBio) and hTIGIT with a K_(D) ofabout 3.80×10⁻¹⁰ M (as determined by MSD-SET), in each case determinedaccording to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 5.26×10⁻¹⁰ M (as determined by ForteBio), cTIGIT with a K_(D) ofabout 7.94×10⁻⁸ M (as determined by ForteBio), and hTIGIT with a K_(D)of about 2.10×10⁻¹⁰ M (as determined by MSD-SET), in each casedetermined according to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 3.78×10⁻¹⁰ M (as determined by ForteBio), cTIGIT with a K_(D) ofabout 7.04×10⁻⁸ M (as determined by ForteBio), and hTIGIT with a K_(D)of about 7.00×10⁻¹¹ M (as determined by MSD-SET), in each casedetermined according to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 4.29×10⁻¹⁰ M (as determined by ForteBio), cTIGIT with a K_(D) ofabout 1.10×10⁻⁷ M (as determined by ForteBio), and hTIGIT with a K_(D)of about 4.10×10⁻¹¹ M (as determined by MSD-SET), in each casedetermined according to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 4.48×10⁻¹⁰ M (as determined by ForteBio) and cTIGIT with a K_(D)of about 7.20×10⁻⁸ M (as determined by ForteBio), in each casedetermined according to the methods provided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 3.00×10⁻¹¹ M, as determined by MSD-SET according to the methodsprovided in Example 4.

In some embodiments, an ABP provided herein binds hTIGIT with a K_(D) ofabout 8.00×10⁻¹¹ M, as determined by MSD-SET according to the methodsprovided in Example 4.

In some embodiments, an ABP provided herein inhibits binding of PVR toTIGIT with an IC₅₀ of about 2.2 nM, about 2.3 nM, about 1.6 nM, about1.9 nM, about 1.7 nM, about 3.2 nM, about 2.6 nM, about 2.9 nM, about3.3 nM, about 2 nM, about 2.2 nM, about 2.1 nM, about 1.8 nM, about 6.4nM, or about 1 nM. In some embodiments, such IC₅₀ ranges from about 1 nMto about 6.4 nM. In some embodiments, such IC₅₀ is about 6.4 nM or less.In some embodiments, such IC₅₀ is determined as described in Example 5.

In some embodiments, an ABP provided herein inhibits binding of PVRL2 toTIGIT with an IC₅₀ of about 1.4 nM, about 1.3 nM, about 1.2 nM, about1.6 nM, about 2 nM, about 1.2 nM, about 1.1 nM, about 1 nM, about 1.8nM, about 1.9 nM, about 2 nM, or about 0.8 nM. In some embodiments, suchIC₅₀ ranges from about 0.8 nM to about 2 nM. In some embodiments, suchIC₅₀ is about 2 nM or less. In some embodiments, such IC₅₀ is determinedas described in Example 5.

In some embodiments, an ABP provided herein inhibits binding of PVR toTIGIT with an IC₅₀ of about 2.2 nM and inhibits binding of PVRL2 toTIGIT with an IC₅₀ of about 1.4 nM. In some embodiments, an ABP providedherein inhibits binding of PVR to TIGIT with an IC₅₀ of about 2.3 nM andinhibits binding of PVRL2 to TIGIT with an IC₅₀ of about 1.3 nM. In someembodiments, an ABP provided herein inhibits binding of PVR to TIGITwith an IC₅₀ of about 1.6 nM and inhibits binding of PVRL2 to TIGIT withan IC₅₀ of about 1.2 nM. In some embodiments, an ABP provided hereininhibits binding of PVR to TIGIT with an IC₅₀ of about 1.9 nM andinhibits binding of PVRL2 to TIGIT with an IC₅₀ of about 1.6 nM. In someembodiments, an ABP provided herein inhibits binding of PVR to TIGITwith an IC₅₀ of about 1.7 nM and inhibits binding of PVRL2 to TIGIT withan IC₅₀ of about 1.4 nM. In some embodiments, an ABP provided hereininhibits binding of PVR to TIGIT with an IC₅₀ of about 3.2 nM andinhibits binding of PVRL2 to TIGIT with an IC₅₀ of about 1.4 nM. In someembodiments, an ABP provided herein inhibits binding of PVR to TIGITwith an IC₅₀ of about 2.6 nM and inhibits binding of PVRL2 to TIGIT withan IC₅₀ of about 2 nM. In some embodiments, an ABP provided hereininhibits binding of PVR to TIGIT with an IC₅₀ of about 2.9 nM andinhibits binding of PVRL2 to TIGIT with an IC₅₀ of about 1.2 nM. In someembodiments, an ABP provided herein inhibits binding of PVR to TIGITwith an IC₅₀ of about 1.9 nM and inhibits binding of PVRL2 to TIGIT withan IC₅₀ of about 1.1 nM. In some embodiments, an ABP provided hereininhibits binding of PVR to TIGIT with an IC₅₀ of about 3.3 nM andinhibits binding of PVRL2 to TIGIT with an IC₅₀ of about 1 nM. In someembodiments, an ABP provided herein inhibits binding of PVR to TIGITwith an IC₅₀ of about 2 nM and inhibits binding of PVRL2 to TIGIT withan IC₅₀ of about 1.2 nM. In some embodiments, an ABP provided hereininhibits binding of PVR to TIGIT with an IC₅₀ of about 1.7 nM andinhibits binding of PVRL2 to TIGIT with an IC₅₀ of about 1.2 nM. In someembodiments, an ABP provided herein inhibits binding of PVR to TIGITwith an IC₅₀ of about 2.1 nM and inhibits binding of PVRL2 to TIGIT withan IC₅₀ of about 1.8 nM. In some embodiments, an ABP provided hereininhibits binding of PVR to TIGIT with an IC₅₀ of about 2.6 nM andinhibits binding of PVRL2 to TIGIT with an IC₅₀ of about 1.6 nM. In someembodiments, an ABP provided herein inhibits binding of PVR to TIGITwith an IC₅₀ of about 2.2 nM and inhibits binding of PVRL2 to TIGIT withan IC₅₀ of about 1.1 nM. In some embodiments, an ABP provided hereininhibits binding of PVR to TIGIT with an IC₅₀ of about 2.1 nM andinhibits binding of PVRL2 to TIGIT with an IC₅₀ of about 1.3 nM. In someembodiments, an ABP provided herein inhibits binding of PVR to TIGITwith an IC₅₀ of about 2.6 nM and inhibits binding of PVRL2 to TIGIT withan IC₅₀ of about 1.9 nM. In some embodiments, an ABP provided hereininhibits binding of PVR to TIGIT with an IC₅₀ of about 1.8 nM andinhibits binding of PVRL2 to TIGIT with an IC₅₀ of about 1.9 nM. In someembodiments, an ABP provided herein inhibits binding of PVR to TIGITwith an IC₅₀ of about 6.4 nM and inhibits binding of PVRL2 to TIGIT withan IC₅₀ of about 2 nM. In some embodiments, an ABP provided hereininhibits binding of PVR to TIGIT with an IC₅₀ of about 2.3 nM andinhibits binding of PVRL2 to TIGIT with an IC₅₀ of about 1.9 nM. In someembodiments, an ABP provided herein inhibits binding of PVR to TIGITwith an IC₅₀ of about 1 nM and inhibits binding of PVRL2 to TIGIT withan IC₅₀ of about 0.8 nM. In some embodiments, such IC₅₀ is about 2 nM orless. In some embodiments, such IC₅₀ is determined as described inExample 5.

2.6.1. Glycosylation Variants

In certain embodiments, an ABP provided herein may be altered toincrease, decrease or eliminate the extent to which it is glycosylated.Glycosylation of polypeptides is typically either “N-linked” or“O-linked.”

“N-linked” glycosylation refers to the attachment of a carbohydratemoiety to the side chain of an asparagine residue. The tripeptidesequences asparagine-X-serine and asparagine-X-threonine, where X is anyamino acid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site.

“O-linked” glycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition or deletion of N-linked glycosylation sites to or from an ABPprovided herein may be accomplished by altering the amino acid sequencesuch that one or more of the above-described tripeptide sequences iscreated or removed. Addition or deletion of O-linked glycosylation sitesmay be accomplished by addition, deletion, or substitution of one ormore serine or threonine residues in or to (as the case may be) thesequence of an ABP.

In some embodiments, an ABP provided herein comprises a glycosylationmotif that is different from a naturally occurring ABP. Any suitablenaturally occurring glycosylation motif can be modified in the ABPsprovided herein. The structural and glycosylation properties ofimmunoglobulins, for example, are known in the art and summarized, forexample, in Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010,125:S41-52, incorporated by reference in its entirety.

In some embodiments, an ABP provided herein comprises an IgG1 Fc regionwith modification to the oligosaccharide attached to asparagine 297 (Asn297). Naturally occurring IgG1 antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn 297 of the C_(H2) domain ofthe Fc region. See Wright et al., TIBTECH, 1997, 15:26-32, incorporatedby reference in its entirety. The oligosaccharide attached to Asn 297may include various carbohydrates such as mannose, N-acetyl glucosamine(GlcNAc), galactose, and sialic acid, as well as a fucose attached to aGlcNAc in the “stem” of the biantennary oligosaccharide structure.

In some embodiments, the oligosaccharide attached to Asn 297 is modifiedto create ABPs having altered ADCC. In some embodiments, theoligosaccharide is altered to improve ADCC. In some embodiments, theoligosaccharide is altered to reduce ADCC.

In some aspects, an ABP provided herein comprises an IgG1 domain withreduced fucose content at position Asn 297 compared to a naturallyoccurring IgG1 domain. Such Fc domains are known to have improved ADCC.See Shields et al., J. Biol. Chem., 2002, 277:26733-26740, incorporatedby reference in its entirety. In some aspects, such ABPs do not compriseany fucose at position Asn 297. The amount of fucose may be determinedusing any suitable method, for example as described in WO 2008/077546,incorporated by reference in its entirety.

In some embodiments, an ABP provided herein comprises a bisectedoligosaccharide, such as a biantennary oligosaccharide attached to theFc region of the ABP that is bisected by GlcNAc. Such ABP variants mayhave reduced fucosylation and/or improved ADCC function. Examples ofsuch ABP variants are described, for example, in WO 2003/011878; U.S.Pat. No. 6,602,684; and U.S. Pat. Pub. No. 2005/0123546; each of whichis incorporated by reference in its entirety.

Other illustrative glycosylation variants which may be incorporated intothe ABPs provided herein are described, for example, in U.S. Pat. Pub.Nos. 2003/0157108, 2004/0093621, 2003/0157108, 2003/0115614,2002/0164328, 2004/0093621, 2004/0132140, 2004/0110704, 2004/0110282,2004/0109865; International Pat. Pub. Nos. 2000/61739, 2001/29246,2003/085119, 2003/084570, 2005/035586, 2005/035778; 2005/053742,2002/031140; Okazaki et al., J. Mol. Biol., 2004, 336:1239-1249; andYamane-Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; each of whichis incorporated by reference in its entirety.

In some embodiments, an ABP provided herein comprises an Fc region withat least one galactose residue in the oligosaccharide attached to the Fcregion. Such ABP variants may have improved CDC function. Examples ofsuch ABP variants are described, for example, in WO 1997/30087; WO1998/58964; and WO 1999/22764; each of which his incorporated byreference in its entirety.

Examples of cell lines capable of producing defucosylated ABPs includeLec13 CHO cells, which are deficient in protein fucosylation (see Ripkaet al., Arch. Biochem. Biophys., 1986, 249:533-545; U.S. Pat. Pub. No.2003/0157108; WO 2004/056312; each of which is incorporated by referencein its entirety), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene or FUT8 knockout CHO cells (seeYamane-Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; Kanda et al.,Biotechnol. Bioeng., 2006, 94:680-688; and WO 2003/085107; each of whichis incorporated by reference in its entirety).

In some embodiments, an ABP provided herein is an aglycosylated ABP. Anaglycosylated ABP can be produced using any method known in the art ordescribed herein. In some aspects, an aglycosylated ABP is produced bymodifying the ABP to remove all glycosylation sites. In some aspects,the glycosylation sites are removed only from the Fc region of the ABP.In some aspects, an aglycosylated ABP is produced by expressing the ABPin an organism that is not capable of glycosylation, such as E. coli, orby expressing the ABP in a cell-free reaction mixture.

In some embodiments, an ABP provided herein has a constant region withreduced effector function compared to a native IgG1 antibody. In someembodiments, the affinity of a constant region of an Fc region of an ABPprovided herein for Fc receptor is less than the affinity of a nativeIgG1 constant region for such Fc receptor.

2.7. Fc Region Amino Acid Sequence Variants

In certain embodiments, an ABP provided herein comprises an Fc regionwith one or more amino acid substitutions, insertions, or deletions incomparison to a naturally occurring Fc region. In some aspects, suchsubstitutions, insertions, or deletions yield ABPs with alteredstability, glycosylation, or other characteristics. In some aspects,such substitutions, insertions, or deletions yield aglycosylated ABPs.

In some aspects, the Fc region of an ABP provided herein is modified toyield an ABP with altered affinity for an Fc receptor, or an ABP that ismore immunologically inert. In some embodiments, the ABP variantsprovided herein possess some, but not all, effector functions. Such ABPsmay be useful, for example, when the half-life of the ABP is importantin vivo, but when certain effector functions (e.g., complementactivation and ADCC) are unnecessary or deleterious.

In some embodiments, the Fc region of an ABP provided herein is a humanIgG4 Fc region comprising one or more of the hinge stabilizing mutationsS228P and L235E. See Aalberse et al., Immunology, 2002, 105:9-19,incorporated by reference in its entirety. In some embodiments, the IgG4Fc region comprises one or more of the following mutations: E233P,F234V, and L235A. See Armour et al., Mol. Immunol., 2003, 40:585-593,incorporated by reference in its entirety. In some embodiments, the IgG4Fc region comprises a deletion at position G236.

In some embodiments, the Fc region of an ABP provided herein is a humanIgG1 Fc region comprising one or more mutations to reduce Fc receptorbinding. In some aspects, the one or more mutations are in residuesselected from 5228 (e.g., S228A), L234 (e.g., L234A), L235 (e.g.,L235A), D265 (e.g., D265A), and N297 (e.g., N297A). In some aspects, theABP comprises a PVA236 mutation. PVA236 means that the amino acidsequence ELLG, from amino acid position 233 to 236 of IgG1 or EFLG ofIgG4, is replaced by PVA. See U.S. Pat. No. 9,150,641, incorporated byreference in its entirety.

In some embodiments, the Fc region of an ABP provided herein is modifiedas described in Armour et al., Eur. J. Immunol., 1999, 29:2613-2624; WO1999/058572; and/or U.K. Pat. App. No. 98099518; each of which isincorporated by reference in its entirety.

In some embodiments, the Fc region of an ABP provided herein is a humanIgG2 Fc region comprising one or more of mutations A330S and P331S.

In some embodiments, the Fc region of an ABP provided herein has anamino acid substitution at one or more positions selected from 238, 265,269, 270, 297, 327 and 329. See U.S. Pat. No. 6,737,056, incorporated byreference in its entirety. Such Fc mutants include Fc mutants withsubstitutions at two or more of amino acid positions 265, 269, 270, 297and 327, including the so-called “DANA” Fc mutant with substitution ofresidues 265 and 297 with alanine. See U.S. Pat. No. 7,332,581,incorporated by reference in its entirety. In some embodiments, the ABPcomprises an alanine at amino acid position 265. In some embodiments,the ABP comprises an alanine at amino acid position 297.

In certain embodiments, an ABP provided herein comprises an Fc regionwith one or more amino acid substitutions which improve ADCC, such as asubstitution at one or more of positions 298, 333, and 334 of the Fcregion. In some embodiments, an ABP provided herein comprises an Fcregion with one or more amino acid substitutions at positions 239, 332,and 330, as described in Lazar et al., Proc. Natl. Acad. Sci. USA, 2006,103:4005-4010, incorporated by reference in its entirety.

In some embodiments, an ABP provided herein comprises one or morealterations that improves or diminishes C1q binding and/or CDC. See U.S.Pat. No. 6,194,551; WO 99/51642; and Idusogie et al., J. Immunol., 2000,164:4178-4184; each of which is incorporated by reference in itsentirety.

In some embodiments, an ABP provided herein comprises one or morealterations to increase half-life. ABPs with increased half-lives andimproved binding to the neonatal Fc receptor (FcRn) are described, forexample, in Hinton et al., J. Immunol., 2006, 176:346-356; and U.S. Pat.Pub. No. 2005/0014934; each of which is incorporated by reference in itsentirety. Such Fc variants include those with substitutions at one ormore of Fc region residues: 238, 250, 256, 265, 272, 286, 303, 305, 307,311, 312, 314, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424,428, and 434 of an IgG.

In some embodiments, an ABP provided herein comprises one or more Fcregion variants as described in U.S. Pat. Nos. 7,371,826, 5,648,260, and5,624,821; Duncan and Winter, Nature, 1988, 322:738-740; and WO94/29351; each of which is incorporated by reference in its entirety.

2.8. Pyroglutamate

As is known in the art, both glutamate (E) and glutamine (Q) at theN-termini of recombinant proteins can cyclize spontaneously to formpyroglutamate (pE) in vitro and in vivo. See Liu et al., J. Biol. Chem.,2011, 286:11211-11217, incorporated by reference in its entirety.

In some embodiments, provided herein are ABPs comprising a polypeptidesequence having a pE residue at the N-terminal position. In someembodiments, provided herein are ABPs comprising a polypeptide sequencein which the N-terminal residue has been converted from Q to pE. In someembodiments, provided herein are ABPs comprising a polypeptide sequencein which the N-terminal residue has been converted from E to pE.

In some embodiments, provided herein are ABPs comprising V_(H) sequenceshaving a pE residue at the N-terminal position. In some embodiments,provided herein are ABPs comprising a V_(H) sequence in which theN-terminal residue has been converted from Q to pE. In some embodiments,provided herein is an ABP comprising a V_(H) sequence selected from SEQID NOs: 4-24, wherein the N-terminal Q residue has been converted to pE.In some embodiments, provided herein is a composition comprising an ABP,wherein the ABP comprises a V_(H) selected from SEQ ID NOs: 4-24, inwhich at least about 20%, at least about 40%, at least about 60%, atleast about 80%, at least about 90%, at least about 95%, or at leastabout 99% of the N-terminal residues of such V_(H) in such compositionhave been converted from Q to pE.

In some embodiments, provided herein are ABPs comprising V_(L) sequenceshaving a pE residue at the N-terminal position. In some embodiments,provided herein are ABPs comprising a V_(L) sequence in which theN-terminal residue has been converted from E to pE. In some embodiments,provided herein is an ABP comprising a V_(L) sequence selected from SEQID NOs: 25-28, wherein the N-terminal E residue has been converted topE. In some embodiments, provided herein is a composition comprising anABP, wherein the ABP comprises a V_(L) selected from SEQ ID NOs: 25-28,in which at least about 20%, at least about 40%, at least about 60%, atleast about 80%, at least about 90%, at least about 95%, or at leastabout 99% of the N-terminal residues of such V_(L) in such compositionhave been converted from E to pE.

In some embodiments, provided herein are ABPs comprising heavy chainsequences having a pE residue at the N-terminal position. In someembodiments, provided herein are ABPs comprising a heavy chain sequencein which the N-terminal residue has been converted from Q to pE. In someembodiments, provided herein is an ABP comprising a heavy chain sequenceselected from SEQ ID NOs: 79, 80, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 123 or124, wherein the N-terminal Q residue has been converted to pE. In someembodiments, provided herein is a composition comprising an ABP, whereinthe ABP comprises a heavy chain selected from SEQ ID NOs: 79, 80, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 121, 122, 123 or 124, in which at least about 20%,at least about 40%, at least about 60%, at least about 80%, at leastabout 90%, at least about 95%, or at least about 99% of the N-terminalresidues of such heavy chain in such composition have been convertedfrom Q to pE.

In some embodiments, provided herein are ABPs comprising light chainsequences having a pE residue at the N-terminal position. In someembodiments, provided herein are ABPs comprising a light chain sequencein which the N-terminal residue has been converted from E to pE. In someembodiments, provided herein is an ABP comprising a light chain sequenceselected from SEQ ID NOs: 81, 92, 107 or 120, wherein the N-terminal Eresidue has been converted to pE. In some embodiments, provided hereinis a composition comprising an ABP, wherein the ABP comprises a lightchain selected from SEQ ID NOs: 81, 92, 107 or 120, in which at leastabout 20%, at least about 40%, at least about 60%, at least about 80%,at least about 90%, at least about 95%, or at least about 99% of theN-terminal residues of such light chain in such composition have beenconverted from E to pE.

2.9. Cysteine Engineered Antigen-Binding Protein Variants

In certain embodiments, provided herein are cysteine engineered ABPs,also known as “thioMAbs,” in which one or more residues of the ABP aresubstituted with cysteine residues. In particular embodiments, thesubstituted residues occur at solvent accessible sites of the ABP. Bysubstituting such residues with cysteine, reactive thiol groups areintroduced at solvent accessible sites of the ABP and may be used toconjugate the ABP to other moieties, such as drug moieties orlinker-drug moieties, for example, to create an immunoconjugate.

In certain embodiments, any one or more of the following residues may besubstituted with cysteine: V205 of the light chain; A118 of the heavychain Fc region; and 5400 of the heavy chain Fc region. Cysteineengineered ABPs may be generated as described, for example, in U.S. Pat.No. 7,521,541, which is incorporated by reference in its entirety.

2.9.1. Immunoconjugates

2.9.1.1. Antigen-Binding Protein-Polymer Conjugates

In some embodiments, an ABP provided herein is derivatized byconjugation with a polymer. Any suitable polymer may be conjugated tothe ABP.

In some embodiments, the polymer is a water soluble polymer.Illustrative examples of water soluble polymers include polyethyleneglycol (PEG), copolymers of ethylene glycol/propylene glycol,carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), poly(n-vinyl pyrrolidone)-co-polyethylene glycol,propropylene glycol homopolymers, polypropylene oxide/ethylene oxideco-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. In some aspects, polyethylene glycolpropionaldehyde may be useful for manufacturing purposes due to itsstability in water.

The polymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to each ABP may vary, and ifmore than one polymer is attached, they may be the same polymer ordifferent polymers. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations includingthe particular properties or functions of the ABP to be improved and theintended use of the ABP.

2.9.1.2. Antigen-Binding Protein-Drug Conjugates

In some embodiments, the ABPs provided herein are conjugated to one ormore therapeutic agents. Any suitable therapeutic agent may beconjugated to the ABP. Exemplary therapeutic agents include cytokines,chemokines, and other agents that induce a desired T cell activity, suchas OX40L, 4-1BBL, TNF-alpha (as used herein, “TNF”), IL-2, IL-15 fusion,CXCL9, CXCL10, IL-10 trap, IL-27 trap, and IL-35 trap. Cytokine trapsand their use are known in the art and described, for example, inEconomides et al., Nature Medicine, 2003, 9:47-52, incorporated byreference in its entirety.

3. Methods of Making TIGIT Antigen-Binding Proteins

3.1. TIGIT Antigen Preparation

The TIGIT antigen used for isolation of the ABPs provided herein may beintact TIGIT or a fragment of TIGIT. The TIGIT antigen may be, forexample, in the form of an isolated protein or a protein expressed onthe surface of a cell.

In some embodiments, the TIGIT antigen is a non-naturally occurringvariant of TIGIT, such as a TIGIT protein having an amino acid sequenceor post-translational modification that does not occur in nature.

In some embodiments, the TIGIT antigen is truncated by removal of, forexample, intracellular or membrane-spanning sequences, or signalsequences. In some embodiments, the TIGIT antigen is fused at itsC-terminus to a human IgG1 Fc domain or a polyhistidine tag.

3.2. Methods of Making Monoclonal Antibodies

Monoclonal antibodies may be obtained, for example, using the hybridomamethod first described by Kohler et al., Nature, 1975, 256:495-497(incorporated by reference in its entirety), and/or by recombinant DNAmethods (see e.g., U.S. Pat. No. 4,816,567, incorporated by reference inits entirety). Monoclonal antibodies may also be obtained, for example,using phage or yeast-based libraries. See e.g., U.S. Pat. Nos. 8,258,082and 8,691,730, each of which is incorporated by reference in itsentirety.

In the hybridoma method, a mouse or other appropriate host animal isimmunized to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein used forimmunization. Alternatively, lymphocytes may be immunized in vitro.Lymphocytes are then fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell. See GodingJ. W., Monoclonal Antibodies: Principles and Practice 3^(rd) ed. (1986)Academic Press, San Diego, Calif., incorporated by reference in itsentirety.

The hybridoma cells are seeded and grown in a suitable culture mediumthat contains one or more substances that inhibit the growth or survivalof the unfused, parental myeloma cells. For example, if the parentalmyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (HATmedium), which substances prevent the growth of HGPRT-deficient cells.

Useful myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive media conditions, such as the presence orabsence of HAT medium. Among these, preferred myeloma cell lines aremurine myeloma lines, such as those derived from MOP-21 and MC-11 mousetumors (available from the Salk Institute Cell Distribution Center, SanDiego, Calif.), and SP-2 or X63-Ag8-653 cells (available from theAmerican Type Culture Collection, Rockville, Md.). Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies. See e.g., Kozbor, J.Immunol., 1984, 133:3001, incorporated by reference in its entirety.

After the identification of hybridoma cells that produce antibodies ofthe desired specificity, affinity, and/or biological activity, selectedclones may be subcloned by limiting dilution procedures and grown bystandard methods. See Goding, supra. Suitable culture media for thispurpose include, for example, D-MEM or RPMI-1640 medium. In addition,the hybridoma cells may be grown in vivo as ascites tumors in an animal.

DNA encoding the monoclonal antibodies may be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the monoclonal antibodies). Thus, thehybridoma cells can serve as a useful source of DNA encoding antibodieswith the desired properties. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asbacteria (e.g., E. coli), yeast (e.g., Saccharomyces or Pichia sp.), COScells, Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce antibody, to produce the monoclonal antibodies.

3.3. Methods of Making Chimeric Antibodies

Illustrative methods of making chimeric antibodies are described, forexample, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.Acad. Sci. USA, 1984, 81:6851-6855; each of which is incorporated byreference in its entirety. In some embodiments, a chimeric antibody ismade by using recombinant techniques to combine a non-human variableregion (e.g., a variable region derived from a mouse, rat, hamster,rabbit, or non-human primate, such as a monkey) with a human constantregion.

3.4. Methods of Making Humanized Antibodies

Humanized antibodies may be generated by replacing most, or all, of thestructural portions of a non-human monoclonal antibody withcorresponding human antibody sequences. Consequently, a hybrid moleculeis generated in which only the antigen-specific variable, or CDR, iscomposed of non-human sequence. Methods to obtain humanized antibodiesinclude those described in, for example, Winter and Milstein, Nature,1991, 349:293-299; Rader et al., Proc. Nat. Acad. Sci. U.S.A., 1998,95:8910-8915; Steinberger et al., J. Biol. Chem., 2000, 275:36073-36078;Queen et al., Proc. Natl. Acad. Sci. U.S.A., 1989, 86:10029-10033; andU.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370; each ofwhich is incorporated by reference in its entirety.

3.5. Methods of Making Human Antibodies

Human antibodies can be generated by a variety of techniques known inthe art, for example by using transgenic animals (e.g., humanized mice).See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. U.S.A., 1993,90:2551; Jakobovits et al., Nature, 1993, 362:255-258; Bruggermann etal., Year in Immuno., 1993, 7:33; and U.S. Pat. Nos. 5,591,669,5,589,369 and 5,545,807; each of which is incorporated by reference inits entirety. Human antibodies can also be derived from phage-displaylibraries (see e.g., Hoogenboom et al., J. Mol. Biol., 1991,227:381-388; Marks et al., J. Mol. Biol., 1991, 222:581-597; and U.S.Pat. Nos. 5,565,332 and 5,573,905; each of which is incorporated byreference in its entirety). Human antibodies may also be generated by invitro activated B cells (see e.g., U.S. Pat. Nos. 5,567,610 and5,229,275, each of which is incorporated by reference in its entirety).Human antibodies may also be derived from yeast-based libraries (seee.g., U.S. Pat. No. 8,691,730, incorporated by reference in itsentirety).

3.6. Methods of Making Antibody Fragments

The antibody fragments provided herein may be made by any suitablemethod, including the illustrative methods described herein or thoseknown in the art. Suitable methods include recombinant techniques andproteolytic digestion of whole antibodies. Illustrative methods ofmaking antibody fragments are described, for example, in Hudson et al.,Nat. Med., 2003, 9:129-134, incorporated by reference in its entirety.Methods of making scFv antibodies are described, for example, inPlückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994);WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458; each of whichis incorporated by reference in its entirety.

3.7. Methods of Making Alternative Scaffolds

The alternative scaffolds provided herein may be made by any suitablemethod, including the illustrative methods described herein or thoseknown in the art. For example, methods of preparing Adnectins™ aredescribed in Emanuel et al., mAbs, 2011, 3:38-48, incorporated byreference in its entirety. Methods of preparing iMabs are described inU.S. Pat. Pub. No. 2003/0215914, incorporated by reference in itsentirety. Methods of preparing Anticalins® are described in Vogt andSkerra, Chem. Biochem., 2004, 5:191-199, incorporated by reference inits entirety. Methods of preparing Kunitz domains are described inWagner et al., Biochem. & Biophys. Res. Comm., 1992, 186:118-1145,incorporated by reference in its entirety. Methods of preparingthioredoxin peptide aptamers are provided in Geyer and Brent, Meth.Enzymol., 2000, 328:171-208, incorporated by reference in its entirety.Methods of preparing Affibodies are provided in Fernandez, Curr. Opinionin Biotech., 2004, 15:364-373, incorporated by reference in itsentirety. Methods of preparing DARPins are provided in Zahnd et al., J.Mol. Biol., 2007, 369:1015-1028, incorporated by reference in itsentirety. Methods of preparing Affilins are provided in Ebersbach etal., J. Mol. Biol., 2007, 372:172-185, incorporated by reference in itsentirety. Methods of preparing Tetranectins are provided in Graversen etal., J. Biol. Chem., 2000, 275:37390-37396, incorporated by reference inits entirety. Methods of preparing Avimers are provided in Silverman etal., Nature Biotech., 2005, 23:1556-1561, incorporated by reference inits entirety. Methods of preparing Fynomers are provided in Silacci etal., J. Biol. Chem., 2014, 289:14392-14398, incorporated by reference inits entirety.

Further information on alternative scaffolds is provided in Binz et al.,Nat. Biotechnol., 2005 23:1257-1268; and Skerra, Current Opin. inBiotech., 2007 18:295-304, each of which is incorporated by reference inits entirety.

3.8. Methods of Making Multispecific ABPs

The multispecific ABPs provided herein may be made by any suitablemethod, including the illustrative methods described herein or thoseknown in the art. Methods of making common light chain antibodies aredescribed in Merchant et al., Nature Biotechnol., 1998, 16:677-681,incorporated by reference in its entirety. Methods of making tetravalentbispecific antibodies are described in Coloma and Morrison, NatureBiotechnol., 1997, 15:159-163, incorporated by reference in itsentirety. Methods of making hybrid immunoglobulins are described inMilstein and Cuello, Nature, 1983, 305:537-540; and Staerz and Bevan,Proc. Natl. Acad. Sci. USA, 1986, 83:1453-1457; each of which isincorporated by reference in its entirety. Methods of makingimmunoglobulins with knobs-into-holes modification are described in U.S.Pat. No. 5,731,168, incorporated by reference in its entirety. Methodsof making immunoglobulins with electrostatic modifications are providedin WO 2009/089004, incorporated by reference in its entirety. Methods ofmaking bispecific single chain antibodies are described in Traunecker etal., EMBO J., 1991, 10:3655-3659; and Gruber et al., J. Immunol., 1994,152:5368-5374; each of which is incorporated by reference in itsentirety. Methods of making single-chain antibodies, whose linker lengthmay be varied, are described in U.S. Pat. Nos. 4,946,778 and 5,132,405,each of which is incorporated by reference in its entirety. Methods ofmaking diabodies are described in Hollinger et al., Proc. Natl. Acad.Sci. USA, 1993, 90:6444-6448, incorporated by reference in its entirety.Methods of making triabodies and tetrabodies are described in Todorovskaet al., J. Immunol. Methods, 2001, 248:47-66, incorporated by referencein its entirety. Methods of making trispecific F(ab′)3 derivatives aredescribed in Tutt et al. J. Immunol., 1991, 147:60-69, incorporated byreference in its entirety. Methods of making cross-linked antibodies aredescribed in U.S. Pat. No. 4,676,980; Brennan et al., Science, 1985,229:81-83; Staerz, et al. Nature, 1985, 314:628-631; and EP 0453082;each of which is incorporated by reference in its entirety. Methods ofmaking antigen-binding domains assembled by leucine zippers aredescribed in Kostelny et al., J. Immunol., 1992, 148:1547-1553,incorporated by reference in its entirety. Methods of making ABPs viathe DNL approach are described in U.S. Pat. Nos. 7,521,056; 7,550,143;7,534,866; and 7,527,787; each of which is incorporated by reference inits entirety. Methods of making hybrids of antibody and non-antibodymolecules are described in WO 93/08829, incorporated by reference in itsentirety, for examples of such ABPs. Methods of making DAF antibodiesare described in U.S. Pat. Pub. No. 2008/0069820, incorporated byreference in its entirety. Methods of making ABPs via reduction andoxidation are described in Carlring et al., PLoS One, 2011, 6:e22533,incorporated by reference in its entirety. Methods of making DVD-Igs™are described in U.S. Pat. No. 7,612,181, incorporated by reference inits entirety. Methods of making DARTs™ are described in Moore et al.,Blood, 2011, 117:454-451, incorporated by reference in its entirety.Methods of making DuoBodies® are described in Labrijn et al., Proc.Natl. Acad. Sci. USA, 2013, 110:5145-5150; Gramer et al., mAbs, 2013,5:962-972; and Labrijn et al., Nature Protocols, 2014, 9:2450-2463; eachof which is incorporated by reference in its entirety. Methods of makingantibodies comprising scFvs fused to the C-terminus of the C_(H3) froman IgG are described in Coloma and Morrison, Nature Biotechnol., 1997,15:159-163, incorporated by reference in its entirety. Methods of makingantibodies in which a Fab molecule is attached to the constant region ofan immunoglobulin are described in Miler et al., J. Immunol., 2003,170:4854-4861, incorporated by reference in its entirety. Methods ofmaking CovX-Bodies are described in Doppalapudi et al., Proc. Natl.Acad. Sci. USA, 2010, 107:22611-22616, incorporated by reference in itsentirety. Methods of making Fcab antibodies are described inWozniak-Knopp et al., Protein Eng. Des. Sel., 2010, 23:289-297,incorporated by reference in its entirety. Methods of making TandAb®antibodies are described in Kipriyanov et al., J. Mol. Biol., 1999,293:41-56 and Zhukovsky et al., Blood, 2013, 122:5116, each of which isincorporated by reference in its entirety. Methods of making tandem Fabsare described in WO 2015/103072, incorporated by reference in itsentirety. Methods of making Zybodies™ are described in LaFleur et al.,mAbs, 2013, 5:208-218, incorporated by reference in its entirety.

3.9. Methods of Making Variants

In some embodiments, an ABP provided herein is an affinity maturedvariant of a parent ABP, which may be generated, for example, usingphage display-based affinity maturation techniques. Briefly, one or moreCDR residues may be mutated and the variant ABPs, or portions thereof,displayed on phage and screened for affinity. Such alterations may bemade in CDR “hotspots,” or residues encoded by codons that undergomutation at high frequency during the somatic maturation process (seeChowdhury, Methods Mol. Biol., 2008, 207:179-196, incorporated byreference in its entirety), and/or residues that contact the antigen.

Any suitable method can be used to introduce variability into apolynucleotide sequence(s) encoding an ABP, including error-prone PCR,chain shuffling, and oligonucleotide-directed mutagenesis such astrinucleotide-directed mutagenesis (TRIM). In some aspects, several CDRresidues (e.g., 4-6 residues at a time) are randomized. CDR residuesinvolved in antigen binding may be specifically identified, for example,using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 inparticular are often targeted for mutation.

The introduction of diversity into the variable regions and/or CDRs canbe used to produce a secondary library. The secondary library is thenscreened to identify ABP variants with improved affinity. Affinitymaturation by constructing and reselecting from secondary libraries hasbeen described, for example, in Hoogenboom et al., Methods in MolecularBiology, 2001, 178:1-37, incorporated by reference in its entirety.

3.10. Vectors, Host Cells, and Recombinant Methods

Also provided are isolated nucleic acids encoding TIGIT ABPs, vectorscomprising the nucleic acids, and host cells comprising the vectors andnucleic acids, as well as recombinant techniques for the production ofthe ABPs.

For recombinant production of an ABP, the nucleic acid(s) encoding itmay be isolated and inserted into a replicable vector for furthercloning (i.e., amplification of the DNA) or expression. In some aspects,the nucleic acid may be produced by homologous recombination, forexample as described in U.S. Pat. No. 5,204,244, incorporated byreference in its entirety.

Many different vectors are known in the art. The vector componentsgenerally include one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence, for example asdescribed in U.S. Pat. No. 5,534,615, incorporated by reference in itsentirety.

Illustrative examples of suitable host cells are provided below. Thesehost cells are not meant to be limiting, and any suitable host cell maybe used to produce the ABPs provided herein.

Suitable host cells include any prokaryotic (e.g., bacterial), lowereukaryotic (e.g., yeast), or higher eukaryotic (e.g., mammalian) cells.Suitable prokaryotes include eubacteria, such as Gram-negative orGram-positive organisms, for example, Enterobacteriaceae such asEscherichia (E. coli), Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella (S. typhimurium), Serratia (S. marcescans), Shigella, Bacilli(B. subtilis and B. licheniformis), Pseudomonas (P. aeruginosa), andStreptomyces. One useful E. coli cloning host is E. coli 294, althoughother strains such as E. coli B, E. coli X1776, and E. coli W3110 arealso suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are also suitable cloning or expression hosts for TIGITABP-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast,is a commonly used lower eukaryotic host microorganism. However, anumber of other genera, species, and strains are available and useful,such as Schizosaccharomyces pombe, Kluyveromyces (K. lactis, K.fragilis, K. bulgaricus K. wickeramii, K. waltii, K. drosophilarum, K.thermotolerans, and K. marxianus), Yarrowia, Pichia pastoris, Candida(C. albicans), Trichoderma reesia, Neurospora crassa, Schwanniomyces (S.occidentalis), and filamentous fungi such as, for example Penicillium,Tolypocladium, and Aspergillus (A. nidulans and A. niger).

Useful mammalian host cells include COS-7 cells, HEK293 cells; babyhamster kidney (BHK) cells; Chinese hamster ovary (CHO); mouse sertolicells; African green monkey kidney cells (VERO-76), and the like.

The host cells used to produce the TIGIT ABP of this invention may becultured in a variety of media. Commercially available media such as,for example, Ham's F10, Minimal Essential Medium (MEM), RPMI-1640, andDulbecco's Modified Eagle's Medium (DMEM) are suitable for culturing thehost cells. In addition, any of the media described in Ham et al., Meth.Enz., 1979, 58:44; Barnes et al., Anal. Biochem., 1980, 102:255; andU.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, and5,122,469; or WO 90/03430 and WO 87/00195 may be used. Each of theforegoing references is incorporated by reference in its entirety.

Any of these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics, trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art.

The culture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

When using recombinant techniques, the ABP can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the ABP is produced intracellularly, as a first step, theparticulate debris, either host cells or lysed fragments, is removed,for example, by centrifugation or ultrafiltration. For example, Carteret al. (Bio/Technology, 1992, 10:163-167, incorporated by reference inits entirety) describes a procedure for isolating ABPs which aresecreted to the periplasmic space of E. coli. Briefly, cell paste isthawed in the presence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris canbe removed by centrifugation.

In some embodiments, the ABP is produced in a cell-free system. In someaspects, the cell-free system is an in vitro transcription andtranslation system as described in Yin et al., mAbs, 2012, 4:217-225,incorporated by reference in its entirety. In some aspects, thecell-free system utilizes a cell-free extract from a eukaryotic cell orfrom a prokaryotic cell. In some aspects, the prokaryotic cell is E.coli. Cell-free expression of the ABP may be useful, for example, wherethe ABP accumulates in a cell as an insoluble aggregate, or where yieldsfrom periplasmic expression are low.

Where the ABP is secreted into the medium, supernatants from suchexpression systems are generally first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon® orMillipore® Pellcon® ultrafiltration unit. A protease inhibitor such asPMSF may be included in any of the foregoing steps to inhibitproteolysis and antibiotics may be included to prevent the growth ofadventitious contaminants.

The ABP composition prepared from the cells can be purified using, forexample, hydroxylapatite chromatography, gel electrophoresis, dialysis,and affinity chromatography, with affinity chromatography being aparticularly useful purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the ABP. Protein A can beused to purify ABPs that comprise human γ1, γ2, or γ4 heavy chains(Lindmark et al., J. Immunol. Meth., 1983, 62:1-13, incorporated byreference in its entirety). Protein G is useful for all mouse isotypesand for human γ3 (Guss et al., EMBO J., 1986, 5:1567-1575, incorporatedby reference in its entirety).

The matrix to which the affinity ligand is attached is most oftenagarose, but other matrices are available. Mechanically stable matricessuch as controlled pore glass or poly(styrenedivinyl)benzene allow forfaster flow rates and shorter processing times than can be achieved withagarose. Where the ABP comprises a C_(H3) domain, the BakerBond ABX®resin is useful for purification.

Other techniques for protein purification, such as fractionation on anion-exchange column, ethanol precipitation, Reverse Phase HPLC,chromatography on silica, chromatography on heparin Sepharose®,chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable, and can be applied by one of skill in the art.

Following any preliminary purification step(s), the mixture comprisingthe ABP of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5 to about 4.5, generally performed at low saltconcentrations (e.g., from about 0 to about 0.25 M salt).

4. Assays

A variety of assays known in the art may be used to identify andcharacterize the TIGIT ABPs provided herein.

4.1. Binding, Competition, and Epitope Mapping Assays

Specific antigen-binding activity of the ABPs provided herein may beevaluated by any suitable method, including using SPR, BLI, RIA andMSD-SET, as described elsewhere in this disclosure. Additionally,antigen-binding activity may be evaluated by ELISA assays and Westernblot assays.

Assays for measuring competition between two ABPs, or an ABP and anothermolecule (e.g., one or more ligands of TIGIT) are described elsewhere inthis disclosure and, for example, in Harlow and Lane, Antibodies: ALaboratory Manual ch. 14, 1988, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., incorporated by reference in its entirety.

Assays for mapping the epitopes to which the ABPs provided herein bindare described, for example, in Morris “Epitope Mapping Protocols,” inMethods in Molecular Biology vol. 66, 1996, Humana Press, Totowa, N.J.,incorporated by reference in its entirety. In some embodiments, theepitope is determined by peptide competition. In some embodiments, theepitope is determined by mass spectrometry. In some embodiments, theepitope is determined by crystallography.

4.2. TIGIT Antagonism Assays

In some embodiments, the ABPs provided herein are screened to identifyor characterize ABPs with antagonistic activity against TIGIT. Anysuitable assay may be used to identify or characterize such ABPs. Insome aspects, the assay measures the amount of a cytokine secreted by aneffector T cell after contacting the effector T cell with an ABPprovided herein. In some aspects, the cytokine is selected from IL-2,IL-6, LT-α, TNF, GM-CSF, IFNγ, and combinations thereof. In someaspects, the cytokine is selected from sCD40L, VEGF, TGF-α, RANTES,PDGF-AB/BB, PDGF-AA, MIP-1β, MIP-1α, MDC (CCL22), MCP-3, MCP-1, IP-10,IL-17A, IL-2Rα, IL-15, IL-13, IL-12 (p70), IL-12 (p40), IL-10, IL-9,IL-8, IL-7, IL-5, IL-4, IL-3, IL-2, IL-2Rα, IL-1RA, IL-1β, IL-1α, IFNγ,IFNα2, GRO, GM-CSF, G-CSF, fractalkine, Flt-3 ligand, FGF-2, eotaxin,EGF, and combinations thereof.

In some embodiments, the effector cells are co-stimulated with anagonist of CD3, to promote the secretion of cytokines by the effectorcell. In some aspects, the CD3 agonist is provided at a submaximallevel.

In some aspects, such assays may measure the proliferation of aneffector T cell after contacting the effector T cell with an ABPprovided herein. In some aspects, proliferation of the effector T cellis measured by dilution of a dye (e.g., carboxyfluorescein diacetatesuccinimidyl ester; CFSE), by tritiated thymidine uptake, by luminescentcell viability assays, or by other assays known in the art.

In some aspects, such assays may measure the differentiation, cytokineproduction, viability (e.g., survival), proliferation, or suppressiveactivity of a regulatory T cell after contacting the regulatory T cellwith an ABP provided herein.

In some aspects, such assays may measure the cytotoxic activity of an NKcell after contacting the NK cell with an ABP provided herein. In someaspects, the cytotoxic activity of the NK cell is measured using acytotoxicity assay that quantifies NK-mediated killing of target cells(e.g., a K562 cell line). See Jang et al., Ann. Clin. Lab. Sci., 2012,42:42-49, incorporated by reference in its entirety.

In some aspects, such assays may measure the amount of granzyme B. Insome aspects, such assays may measure the amount of perforin.

4.3. Assays for Effector Functions

Effector function following treatment with the ABPs provided herein maybe evaluated using a variety of in vitro and in vivo assays known in theart, including those described in Ravetch and Kinet, Annu. Rev.Immunol., 1991, 9:457-492; U.S. Pat. Nos. 5,500,362, 5,821,337;Hellstrom et al., Proc. Nat'l Acad. Sci. USA, 1986, 83:7059-7063;Hellstrom et al., Proc. Nat'l Acad. Sci. USA, 1985, 82:1499-1502;Bruggemann et al., J. Exp. Med., 1987, 166:1351-1361; Clynes et al.,Proc. Nat'l Acad. Sci. USA, 1998, 95:652-656; WO 2006/029879; WO2005/100402; Gazzano-Santoro et al., J. Immunol. Methods, 1996,202:163-171; Cragg et al., Blood, 2003, 101:1045-1052; Cragg et al.Blood, 2004, 103:2738-2743; and Petkova et al., Int'l. Immunol., 2006,18:1759-1769; each of which is incorporated by reference in itsentirety.

5. Pharmaceutical Compositions

The ABPs provided herein can be formulated in any appropriatepharmaceutical composition and administered by any suitable route ofadministration. Suitable routes of administration include, but are notlimited to, the intraarterial, intradermal, intramuscular,intraperitoneal, intravenous, nasal, parenteral, pulmonary, andsubcutaneous routes.

The pharmaceutical composition may comprise one or more pharmaceuticalexcipients. Any suitable pharmaceutical excipient may be used, and oneof ordinary skill in the art is capable of selecting suitablepharmaceutical excipients. Accordingly, the pharmaceutical excipientsprovided below are intended to be illustrative, and not limiting.Additional pharmaceutical excipients include, for example, thosedescribed in the Handbook of Pharmaceutical Excipients, Rowe et al.(Eds.) 6th Ed. (2009), incorporated by reference in its entirety.

In some embodiments, the pharmaceutical composition comprises ananti-foaming agent. Any suitable anti-foaming agent may be used. In someaspects, the anti-foaming agent is selected from an alcohol, an ether,an oil, a wax, a silicone, a surfactant, and combinations thereof. Insome aspects, the anti-foaming agent is selected from a mineral oil, avegetable oil, ethylene bis stearamide, a paraffin wax, an ester wax, afatty alcohol wax, a long chain fatty alcohol, a fatty acid soap, afatty acid ester, a silicon glycol, a fluorosilicone, a polyethyleneglycol-polypropylene glycol copolymer, polydimethylsiloxane-silicondioxide, ether, octyl alcohol, capryl alcohol, sorbitan trioleate, ethylalcohol, 2-ethyl-hexanol, dimethicone, oleyl alcohol, simethicone, andcombinations thereof.

In some embodiments, the pharmaceutical composition comprises acosolvent. Illustrative examples of cosolvents include ethanol,poly(ethylene) glycol, butylene glycol, dimethylacetamide, glycerin,propylene glycol, and combinations thereof.

In some embodiments, the pharmaceutical composition comprises a buffer.Illustrative examples of buffers include acetate, borate, carbonate,lactate, malate, phosphate, citrate, hydroxide, diethanolamine,monoethanolamine, glycine, methionine, guar gum, monosodium glutamate,and combinations thereof.

In some embodiments, the pharmaceutical composition comprises a carrieror filler. Illustrative examples of carriers or fillers include lactose,maltodextrin, mannitol, sorbitol, chitosan, stearic acid, xanthan gum,guar gum, and combinations thereof.

In some embodiments, the pharmaceutical composition comprises asurfactant. Illustrative examples of surfactants include d-alphatocopherol, benzalkonium chloride, benzethonium chloride, cetrimide,cetylpyridinium chloride, docusate sodium, glyceryl behenate, glycerylmonooleate, lauric acid, macrogol 15 hydroxystearate, myristyl alcohol,phospholipids, polyoxyethylene alkyl ethers, polyoxyethylene sorbitanfatty acid esters, polyoxyethylene stearates, polyoxylglycerides, sodiumlauryl sulfate, sorbitan esters, vitamin E polyethylene(glycol)succinate, and combinations thereof.

In some embodiments, the pharmaceutical composition comprises ananti-caking agent. Illustrative examples of anti-caking agents includecalcium phosphate (tribasic), hydroxymethyl cellulose, hydroxypropylcellulose, magnesium oxide, and combinations thereof.

Other excipients that may be used with the pharmaceutical compositionsinclude, for example, albumin, antioxidants, antibacterial agents,antifungal agents, bioabsorbable polymers, chelating agents, controlledrelease agents, diluents, dispersing agents, dissolution enhancers,emulsifying agents, gelling agents, ointment bases, penetrationenhancers, preservatives, solubilizing agents, solvents, stabilizingagents, sugars, and combinations thereof. Specific examples of each ofthese agents are described, for example, in the Handbook ofPharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), ThePharmaceutical Press, incorporated by reference in its entirety.

In some embodiments, the pharmaceutical composition comprises a solvent.In some aspects, the solvent is saline solution, such as a sterileisotonic saline solution or dextrose solution. In some aspects, thesolvent is water for injection.

In some embodiments, the pharmaceutical compositions are in aparticulate form, such as a microparticle or a nanoparticle.Microparticles and nanoparticles may be formed from any suitablematerial, such as a polymer or a lipid. In some aspects, themicroparticles or nanoparticles are micelles, liposomes, orpolymersomes.

Further provided herein are anhydrous pharmaceutical compositions anddosage forms comprising an ABP, since water can facilitate thedegradation of some ABPs.

Anhydrous pharmaceutical compositions and dosage forms provided hereincan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine can be anhydrousif substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions can be packaged using materials known to prevent exposureto water such that they can be included in suitable formulary kits.Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs.

5.1. Parenteral Dosage Forms

In certain embodiments, the ABPs provided herein are formulated asparenteral dosage forms. Parenteral dosage forms can be administered tosubjects by various routes including, but not limited to, subcutaneous,intravenous (including infusions and bolus injections), intramuscular,and intraarterial. Because their administration typically bypassessubjects' natural defenses against contaminants, parenteral dosage formsare typically, sterile or capable of being sterilized prior toadministration to a subject. Examples of parenteral dosage formsinclude, but are not limited to, solutions ready for injection, dry(e.g., lyophilized) products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage formsare well known to those skilled in the art. Examples include, but arenot limited to: Water for Injection USP; aqueous vehicles such as, butnot limited to, Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, and Lactated Ringer'sInjection; water miscible vehicles such as, but not limited to, ethylalcohol, polyethylene glycol, and polypropylene glycol; and non-aqueousvehicles such as, but not limited to, corn oil, cottonseed oil, peanutoil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Excipients that increase the solubility of one or more of the ABPsdisclosed herein can also be incorporated into the parenteral dosageforms.

In some embodiments, the parenteral dosage form is lyophilized.Exemplary lyophilized formulations are described, for example, in U.S.Pat. Nos. 6,267,958 and 6,171,586; and WO 2006/044908; each of which isincorporated by reference in its entirety.

6. Dosage and Unit Dosage Forms

In human therapeutics, the doctor will determine the posology which heconsiders most appropriate according to a preventive or curativetreatment and according to the age, weight, condition and other factorsspecific to the subject to be treated.

In certain embodiments, a composition provided herein is apharmaceutical composition or a single unit dosage form. Pharmaceuticalcompositions and single unit dosage forms provided herein comprise aprophylactically or therapeutically effective amount of one or moreprophylactic or therapeutic ABPs.

The amount of the ABP or composition which will be effective in theprevention or treatment of a disorder or one or more symptoms thereofwill vary with the nature and severity of the disease or condition, andthe route by which the ABP is administered. The frequency and dosagewill also vary according to factors specific for each subject dependingon the specific therapy (e.g., therapeutic or prophylactic agents)administered, the severity of the disorder, disease, or condition, theroute of administration, as well as age, body, weight, response, and thepast medical history of the subject. Effective doses may be extrapolatedfrom dose-response curves derived from in vitro or animal model testsystems.

In certain embodiments, exemplary doses of a composition includemilligram or microgram amounts of the ABP per kilogram of subject orsample weight (e.g., about 10 micrograms per kilogram to about 50milligrams per kilogram, about 100 micrograms per kilogram to about 25milligrams per kilogram, or about 100 microgram per kilogram to about 10milligrams per kilogram). In certain embodiment, the dosage of the ABPprovided herein, based on weight of the ABP, administered to prevent,treat, manage, or ameliorate a disorder, or one or more symptoms thereofin a subject is 0.1 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg,6 mg/kg, 10 mg/kg, or 15 mg/kg or more of a subject's body weight. Itmay be necessary to use dosages of the ABP outside the ranges disclosedherein in some cases, as will be apparent to those of ordinary skill inthe art. Furthermore, it is noted that the clinician or treatingphysician will know how and when to interrupt, adjust, or terminatetherapy in conjunction with subject response.

Different therapeutically effective amounts may be applicable fordifferent diseases and conditions, as will be readily known by those ofordinary skill in the art. Similarly, amounts sufficient to prevent,manage, treat or ameliorate such disorders, but insufficient to cause,or sufficient to reduce, adverse effects associated with the ABPsprovided herein are also encompassed by the dosage amounts and dosefrequency schedules provided herein. Further, when a subject isadministered multiple dosages of a composition provided herein, not allof the dosages need be the same. For example, the dosage administered tothe subject may be increased to improve the prophylactic or therapeuticeffect of the composition or it may be decreased to reduce one or moreside effects that a particular subject is experiencing.

In certain embodiments, treatment or prevention can be initiated withone or more loading doses of an ABP or composition provided hereinfollowed by one or more maintenance doses.

In certain embodiments, a dose of an ABP or composition provided hereincan be administered to achieve a steady-state concentration of the ABPin blood or serum of the subject. The steady-state concentration can bedetermined by measurement according to techniques available to those ofskill or can be based on the physical characteristics of the subjectsuch as height, weight and age.

In certain embodiments, administration of the same composition may berepeated and the administrations may be separated by at least 1 day, 2days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75days, 3 months, or 6 months.

As discussed in more detail elsewhere in this disclosure, an ABPprovided herein may optionally be administered with one or moreadditional agents useful to prevent or treat a disease or disorder. Theeffective amount of such additional agents may depend on the amount ofABP present in the formulation, the type of disorder or treatment, andthe other factors known in the art or described herein.

7. Therapeutic Applications

For therapeutic applications, the ABPs of the invention are administeredto a mammal, generally a human, in a pharmaceutically acceptable dosageform such as those known in the art and those discussed above. Forexample, the ABPs of the invention may be administered to a humanintravenously as a bolus or by continuous infusion over a period oftime, by intramuscular, intraperitoneal, intra-cerebrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, orintratumoral routes. The ABPs also are suitably administered byperitumoral, intralesional, or perilesional routes, to exert local aswell as systemic therapeutic effects. The intraperitoneal route may beparticularly useful, for example, in the treatment of ovarian tumors.

The ABPs provided herein may be useful for the treatment of any diseaseor condition involving TIGIT. In some embodiments, the disease orcondition is a disease or condition that can benefit from treatment withan anti-TIGIT ABP. In some embodiments, the disease or condition is atumor. In some embodiments, the disease or condition is a cellproliferative disorder. In some embodiments, the disease or condition isa cancer. In some embodiments, the disease or condition is a viralinfection.

In some embodiments, the ABPs provided herein are provided for use as amedicament. In some embodiments, the ABPs provided herein are providedfor use in the manufacture or preparation of a medicament. In someembodiments, the medicament is for the treatment of a disease orcondition that can benefit from an anti-TIGIT ABP. In some embodiments,the disease or condition is a tumor. In some embodiments, the disease orcondition is a cell proliferative disorder. In some embodiments, thedisease or condition is a cancer. In some embodiments, the disease orcondition is a viral infection.

In some embodiments, provided herein is a method of treating a diseaseor condition in a subject in need thereof by administering an effectiveamount of an ABP provided herein to the subject. In some aspects, thedisease or condition is a cancer. In some aspects, the disease orcondition is a viral infection.

Any suitable cancer may be treated with the ABPs provided herein.Illustrative suitable cancers include, for example, acute lymphoblasticleukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma,anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, braintumor, bile duct cancer, bladder cancer, bone cancer, breast cancer,bronchial tumor, carcinoma of unknown primary origin, cardiac tumor,cervical cancer, chordoma, colon cancer, colorectal cancer,craniopharyngioma, ductal carcinoma, embryonal tumor, endometrialcancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibroushistiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladdercancer, gastric cancer, gastrointestinal carcinoid tumor,gastrointestinal stromal tumor, gestational trophoblastic disease,glioma, head and neck cancer, hepatocellular cancer, histiocytosis,Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, isletcell tumor, Kaposi sarcoma, kidney cancer, Langerhans cellhistiocytosis, laryngeal cancer, lip and oral cavity cancer, livercancer, lobular carcinoma in situ, lung cancer, macroglobulinemia,malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma,mesothelioma, metastatic squamous neck cancer with occult primary,midline tract carcinoma involving NUT gene, mouth cancer, multipleendocrine neoplasia syndrome, multiple myeloma, mycosis fungoides,myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasm,nasal cavity and par nasal sinus cancer, nasopharyngeal cancer,neuroblastoma, non-small cell lung cancer, oropharyngeal cancer,osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis,paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer,pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primarycentral nervous system lymphoma, prostate cancer, rectal cancer, renalcell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoidtumor, salivary gland cancer, Sezary syndrome, skin cancer, small celllung cancer, small intestine cancer, soft tissue sarcoma, spinal cordtumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicularcancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer,urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, andWilms tumor.

Any suitable virus may be treated with the ABPs provided herein.Illustrative suitable viruses include, for example, adeno-associatedvirus, Aichi virus, Australian bat lyssavirus, BK polyomavirus, Bannavirus, Barmah forest virus, Bunyamwera virus, Bunyavirus La Crosse,Bunyavirus snowshoe hare, Cercopithecine herpesvirus, Chandipura virus,Chikungunya virus, Cosavirus A, cowpox virus, Coxsackievirus,Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhori virus, Dugbevirus, Duvenhage virus, eastern equine encephalitis virus, ebolavirus,echovirus, encephalomyocarditis virus, Epstein-Barr virus, European batlyssavirus, GB virus C/Hepatitis G virus, Hantaan virus, Hendra virus,hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis Evirus, hepatitis delta virus, horsepox virus, human adenovirus, humanastrovirus, human coronavirus, human cytomegalovirus, human enterovirus,human herpesvirus 1, human herpesvirus 2, human herpesvirus 6, humanherpesvirus 7, human herpesvirus 8, human immunodeficiency virus, humanpapillomavirus 1, human papillomavirus 2, human papillomavirus, humanparainfluenza, human parvovirus B19, human respiratory syncytial virus,human rhinovirus, human SARS coronavirus, human spumaretrovirus, humanT-lymphotropic virus, human torovirus, influenza A virus, influenza Bvirus, influenza C virus, Isfahan virus, JC polyomavirus, Japaneseencephalitis virus, Junin arenavirus, KI polyomavirus, Kunjin virus,Lagos bat virus, Lake Victoria marburgvirus, Langat virus, Lassa virus,Lordsdale virus, Louping ill virus, lymphocytic choriomeningitis virus,Machupo virus, Mayaro virus, MERS coronavirus, measles virus, Mengoencephalomyocarditis virus, Merkel cell polyomavirus, Mokola virus,molluscum contagiosum virus, monkeypox virus, mumps virus, Murray valleyencephalitis virus, New York virus, Nipah virus, Norwalk virus,O'nyong-nyong virus, Orf virus, Oropouche virus, Pichinde virus,poliovirus, Punta toro phlebovirus, Puumala virus, rabies virus, RiftValley fever virus, Rosavirus A, Ross River virus, rotavirus A,rotavirus B, rotavirus C, rubella virus, Sagiyama virus, salivirus A,sandfly fever Sicilian virus, Sapporo virus, Semliki Forest virus, Seoulvirus, simian foamy virus, simian virus 5, Sindbis virus, Southamptonvirus, St. Louis encephalitis virus, tick-borne powassan virus, torqueteno virus, Toscana virus, Uukuniemi virus, vaccinia virus,varicella-zoster virus, variola virus, Venezuelan equine encephalitisvirus, vesicular stomatitis virus, western equine encephalitis virus, WUpolyomavirus, West Nile virus, Yaba monkey tumor virus, Yaba-likedisease virus, yellow fever virus, and Zika virus.

In some embodiments, provided herein is a method of antagonizing TIGITin a target cell of a subject in need thereof by administering aneffective amount of an ABP provided herein to the subject. In someaspects, antagonism of TIGIT by an ABP provided herein results inincreased secretion of IL-2, LT-α, IL-6, TNF, GM-CSF, IFNγ orcombinations thereof by a target cell.

In some embodiments, provided herein is a method of increasing theproliferation, survival, and/or function of an effector T cell in asubject in need thereof by administering an effective amount of an ABPprovided herein to the subject. In some aspects the effector T cell is aCD4+ effector T cell. In some aspects, the effector T cell is a CD8+effector T cell.

In some embodiments, provided herein is a method of abrogatingsuppression of an effector T cell by a regulatory T cell in a subject inneed thereof by administering an effective amount of an ABP providedherein to the subject. In some aspects, the regulatory T cell is aCD4+CD25+Foxp3+ regulator T cell. In some aspects, the regulatory T cellis a CD8+CD25+ regulatory T cell.

In some embodiments, provided herein is a method of increasing theactivity of a natural killer (NK) or natural killer T (NKT) cell in asubject in need thereof by administering an effective amount of an ABPprovided herein to the subject.

In some embodiments, provided herein is a method of enhancing an immuneresponse in a subject in need thereof by administering an effectiveamount of an ABP provided herein to the subject.

In some embodiments, provided herein is a method delaying the onset of atumor in a subject in need thereof by administering an effective amountof an ABP provided herein to the subject.

In some embodiments, provided herein is a method preventing the onset ofa tumor in a subject in need thereof by administering an effectiveamount of an ABP provided herein to the subject.

In some embodiments, provided herein is a method of delaying the onsetof a cancer in a subject in need thereof by administering an effectiveamount of an ABP provided herein to the subject.

In some embodiments, provided herein is a method of preventing the onsetof a cancer in a subject in need thereof by administering an effectiveamount of an ABP provided herein to the subject.

In some embodiments, provided herein is a method of reducing the size ofa tumor in a subject in need thereof by administering an effectiveamount of an ABP provided herein to the subject.

In some embodiments, provided herein is a method of reducing the numberof metastases in a subject in need thereof by administering an effectiveamount of an ABP provided herein to the subject.

In some embodiments, provided herein is a method of delaying the onsetof a viral infection in a subject in need thereof by administering aneffective amount of an ABP provided herein to the subject.

In some embodiments, provided herein is a method of preventing the onsetof a viral infection in a subject in need thereof by administering aneffective amount of an ABP provided herein to the subject.

In some embodiments, provided herein is a method of reducing viral titera subject in need thereof by administering an effective amount of an ABPprovided herein to the subject.

In some embodiments, provided herein is a method of eliminating a virusfrom subject in need thereof by administering an effective amount of anABP provided herein to the subject.

In some embodiments, provided herein is a method for extending theperiod of overall survival, median survival time, or progression-freesurvival in a subject in need thereof by administering an effectiveamount of an ABP provided herein to the subject.

In some embodiments, provided herein is a method for treating a subjectwho has become resistant to a standard of care therapeutic byadministering an effective amount of an ABP provided herein to thesubject. In some embodiments, the standard-of-care therapeutic to whichthe subject has become resistant is a PD-1 inhibitor. In otherembodiments, the standard-of-care therapeutic to which the subject hasbecome resistant is a PD-L1 inhibitor. In other embodiments, thestandard-of-care therapeutic to which the subject has become resistantis a CTLA-4 inhibitor.

8. Combination Therapies

In some embodiments, an ABP provided herein is administered with atleast one additional therapeutic agent. Any suitable additionaltherapeutic agent may be administered with an ABP provided herein. Insome aspects, the additional therapeutic agent is selected fromradiation, a cytotoxic agent, a chemotherapeutic agent, a cytostaticagent, an anti-hormonal agent, an EGFR inhibitor, an immunostimulatoryagent, an anti-angiogenic agent, and combinations thereof.

In some embodiments, the additional therapeutic agent comprises animmunostimulatory agent.

In some embodiments, the immunostimulatory agent is an agent that blockssignaling of an inhibitory receptor of an immune cell, or a ligandthereof. In some aspects, the inhibitory receptor or ligand is selectedfrom CTLA-4, PD-1, PD-L1, LAG-3, Tim3, TIGIT, neuritin, BTLA, KIR, andcombinations thereof. In some aspects, the agent is selected from ananti-PD-1 antibody (e.g., pembrolizumab or nivolumab), and anti-PD-L1antibody (e.g., atezolizumab), an anti-CTLA-4 antibody (e.g.,ipilimumab), and combinations thereof. In some aspects, the agent ispembrolizumab. In some aspects, the agent is nivolumab. In some aspects,the agent is atezolizumab.

In some embodiments, the additional therapeutic agent is an agent thatinhibits the interaction between PD-1 and PD-L1. In some aspects, theadditional therapeutic agent that inhibits the interaction between PD-1and PD-L1 is selected from an antibody, a peptidomimetic and a smallmolecule. In some aspects, the additional therapeutic agent thatinhibits the interaction between PD-1 and PD-L1 is selected frompembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab,BMS-936559, sulfamonomethoxine 1, and sulfamethizole 2. In someembodiments, the additional therapeutic agent that inhibits theinteraction between PD-1 and PD-L1 is any therapeutic known in the artto have such activity, for example as described in Weinmann et al., ChemMed Chem, 2016, 14:1576 (DOI: 10.1002/cmdc.201500566), incorporated byreference in its entirety. In some embodiments, the agent that inhibitsthe interaction between PD-1 and PD-L1 is formulated in the samepharmaceutical composition an ABP provided herein. In some embodiments,the agent that inhibits the interaction between PD-1 and PD-L1 isformulated in a different pharmaceutical composition from an ABPprovided herein. In some embodiments, the agent that inhibits theinteraction between PD-1 and PD-L1 is administered prior toadministration of an ABP provided herein. In some embodiments, the agentthat inhibits the interaction between PD-1 and PD-L1 is administeredafter administration of an ABP provided herein. In some embodiments, theagent that inhibits the interaction between PD-1 and PD-L1 isadministered contemporaneously with an ABP provided herein, but theagent and ABP are administered in separate pharmaceutical compositions.

In some embodiments, the immunostimulatory agent is an agonist of aco-stimulatory receptor of an immune cell. In some aspects, theco-stimulatory receptor is selected from OX40, ICOS, CD27, CD28, 4-1BB,or CD40. In some embodiments, the agonist is an antibody.

In some embodiments, the immunostimulatory agent is a cytokine. In someaspects, the cytokine is selected from IL-2, IL-5, IL-7, IL-12, IL-15,IL-21, and combinations thereof.

In some embodiments, the immunostimulatory agent is an oncolytic virus.In some aspects, the oncolytic virus is selected from a herpes simplexvirus, a vesicular stomatitis virus, an adenovirus, a Newcastle diseasevirus, a vaccinia virus, and a maraba virus.

In some embodiments, the immunostimulatory agent is a T cell with achimeric antigen receptor (CAR-T cell). In some embodiments, theimmunostimulatory agent is a bi- or multi-specific T cell directedantibody. In some embodiments, the immunostimulatory agent is ananti-TGF-β antibody. In some embodiments, the immunostimulatory agent isa TGF-β trap.

In some embodiments, the additional therapeutic agent is a vaccine to atumor antigen. Any suitable antigen may be targeted by the vaccine,provided that it is present in a tumor treated by the methods providedherein. In some aspects, the tumor antigen is a tumor antigen that isoverexpressed in comparison its expression levels in normal tissue. Insome aspects, the tumor antigen is selected from cancer testis antigen,differentiation antigen, NY-ESO-1, MAGE-A1, MART, and combinationsthereof.

Further examples of additional therapeutic agents include a taxane(e.g., paclitaxel or docetaxel); a platinum agent (e.g., carboplatin,oxaliplatin, and/or cisplatin); a topoisomerase inhibitor (e.g.,irinotecan, topotecan, etoposide, and/or mitoxantrone); folinic acid(e.g., leucovorin); or a nucleoside metabolic inhibitor (e.g.,fluorouracil, capecitabine, and/or gemcitabine). In some embodiments,the additional therapeutic agent is folinic acid, 5-fluorouracil, and/oroxaliplatin. In some embodiments, the additional therapeutic agent is5-fluorouracil and irinotecan. In some embodiments, the additionaltherapeutic agent is a taxane and a platinum agent. In some embodiments,the additional therapeutic agent is paclitaxel and carboplatin. In someembodiments, the additional therapeutic agent is pemetrexate. In someembodiments, the additional therapeutic agent is a targeted therapeuticsuch as an EGFR, RAF or MEK-targeted agent.

The additional therapeutic agent may be administered by any suitablemeans. In some embodiments, an ABP provided herein and the additionaltherapeutic agent are included in the same pharmaceutical composition.In some embodiments, an ABP provided herein and the additionaltherapeutic agent are included in different pharmaceutical compositions.

In embodiments where an ABP provided herein and the additionaltherapeutic agent are included in different pharmaceutical compositions,administration of the ABP can occur prior to, simultaneously, and/orfollowing, administration of the additional therapeutic agent. In someaspects, administration of an ABP provided herein and the additionaltherapeutic agent occur within about one month of each other. In someaspects, administration of an ABP provided herein and the additionaltherapeutic agent occur within about one week of each other. In someaspects, administration of an ABP provided herein and the additionaltherapeutic agent occur within about one day of each other. In someaspects, administration of an ABP provided herein and the additionaltherapeutic agent occur within about twelve hours of each other. In someaspects, administration of an ABP provided herein and the additionaltherapeutic agent occur within about one hour of each other.

9. Diagnostic Methods

Also provided are methods for detecting the presence of TIGIT on cellsfrom a subject. Such methods may be used, for example, to predict andevaluate responsiveness to treatment with an ABP provided herein.

In some embodiments, a blood sample is obtained from a subject and thefraction of cells expressing TIGIT is determined. In some aspects, therelative amount of TIGIT expressed by such cells is determined. Thefraction of cells expressing TIGIT and the relative amount of TIGITexpressed by such cells can be determined by any suitable method. Insome embodiments, flow cytometry is used to make such measurements. Insome embodiments, fluorescence assisted cell sorting (FACS) is used tomake such measurement. See Li et al., J. Autoimmunity, 2003, 21:83-92for methods of evaluating expression of TIGIT in peripheral blood.

10. Kits

Also provided are kits comprising the ABPs provided herein. The kits maybe used for the treatment, prevention, and/or diagnosis of a disease ordisorder, as described herein.

In some embodiments, the kit comprises a container and a label orpackage insert on or associated with the container. Suitable containersinclude, for example, bottles, vials, syringes, and IV solution bags.The containers may be formed from a variety of materials, such as glassor plastic. The container holds a composition that is by itself, or whencombined with another composition, effective for treating, preventingand/or diagnosing a disease or disorder. The container may have asterile access port. For example, if the container is an intravenoussolution bag or a vial, it may have a port that can be pierced by aneedle. At least one active agent in the composition is an ABP providedherein. The label or package insert indicates that the composition isused for treating the selected condition.

In some embodiments, the kit comprises (a) a first container with afirst composition contained therein, wherein the first compositioncomprises an ABP provided herein; and (b) a second container with asecond composition contained therein, wherein the second compositioncomprises a further therapeutic agent. The kit in this embodiment of theinvention may further comprise a package insert indicating that thecompositions can be used to treat a particular condition.

Alternatively, or additionally, the kit may further comprise a second(or third) container comprising a pharmaceutically-acceptable excipient.In some aspects, the excipient is a buffer. The kit may further includeother materials desirable from a commercial and user standpoint,including filters, needles, and syringes.

11. Other Illustrative Embodiments

The embodiments provided below are non-limiting and provided by way ofillustration of certain embodiments and aspects of the invention, inaddition to those described throughout this disclosure.

Embodiment 1

An antigen binding protein that binds specifically to a human TIGIT(hTIGIT) and is capable of at least one of the following: a) inhibitsbinding of hTIGIT to CD155 and CD112; b) increases a T effector cellfunction; c) increases a natural killer (NK) cell function; d) decreasesthe number of regulatory T cells in tissues or in circulation; e)suppresses a regulatory T cell or a regulatory T cell activity; f)inhibits association of TIGIT and CD226; and does not bind specificallyto Nectin-4 (also known as poliovirus-receptor-like 4, PVRL4).

Embodiment 2

The antigen binding protein of Embodiment 1, wherein the antigen bindingprotein has one or more of the following characteristics: a) is amonoclonal antibody; b) is a human antibody, a humanized antibody, or achimeric antibody; c) is a bispecific antibody, a multispecificantibody, a diabody, or a multivalent antibody; d) is of the IgG1, IgG2,IgG3, the IgG4 type, or the IgG4 isotype with a S228P substitution; e)is an antigen-binding antibody fragment; f) is a Fab fragment, a Fab′fragment, a F(ab′)2 fragment, or an Fv fragment; g) is a single chainantibody, a single domain antibody, or a nanobody.

Embodiment 3

A pharmaceutical composition comprising an effective amount of anantibody which binds to hTIGIT and: (a) increases cell-mediatedimmunity; (b) increases T-cell activity; (c) increases cytolytic T-cell(CTL) activity; (d) increases natural killer (NK) cell activity; (e) isan antagonist of TIGIT-mediated signaling; (f) inhibits TIGIT signaling;(g) inhibits or blocks the interaction between PVR and TIGIT; (h)inhibits or blocks the interaction of TIGIT and CD155 ligand and/orCD112; but does not inhibit the interaction between PVR and CD226.

Embodiment 4

A pharmaceutical composition comprising the antigen-binding protein ofEmbodiment 1 or Embodiment 2.

Embodiment 5

The pharmaceutical composition of Embodiment 4, further comprising aneffective amount of an anti-PD-1 antibody.

Embodiment 6

The antigen binding protein of Embodiment 1, wherein the antigen bindingprotein has one or more of the following characteristics: a) binds to ahuman TIGIT polypeptide or a variant thereof, or as otherwise providedherein with a K_(D) of less than about 20 nM; or b) binds to acynomolgus monkey (also “cynomolgus” or “cyno”) TIGIT polypeptide or avariant thereof, or as otherwise provided herein, with a K_(D) of lessthan about 200 nM; c) binds to a murine TIGIT polypeptide or a variantthereof, or as otherwise provided herein, with a K_(D) of less thanabout 200 nM; or d) a combination of at least 2 of a), b), and c).

Embodiment 7

An antigen binding protein that competes or is capable of competing forbinding to human TIGIT with a reference antigen binding protein, whereinthe reference antigen binding protein is the antigen binding protein ofEmbodiment 1.

Embodiment 8

The antigen binding protein of Embodiment 7, wherein the antigen bindingprotein and the reference antibody cross-compete or are capable ofcross-competing for binding to a human TIGIT.

Embodiment 9

The antigen binding protein of Embodiment 1, comprising a heavy chainconstant region comprising a human heavy chain constant region orfragment or a variant thereof, wherein the constant region variantcomprises up to 20 conservatively modified amino acid substitutions.

Embodiment 10

The antigen binding protein of Embodiment 1, that competes or is capableof competing for binding to human TIGIT with a CD155 protein and/or aCD112 protein.

Embodiment 11

The antigen binding protein of Embodiment 1, that is capable ofantagonizing TIGIT signaling in a T cell-specific manner.

Embodiment 12

An isolated antibody molecule capable of binding to human TIGIT(hTIGIT), comprising a heavy chain variable region (VH) comprising aVHCDR1 amino acid sequence of SEQ ID NOs: 48-62, a VHCDR2 amino acidsequence of SEQ ID NO:36-47, and a VHCDR3 amino acid sequence of SEQ IDNO:29-35; and a light chain variable region (VL) comprising a VLCDR1amino acid sequence of SEQ ID NO:70-72, a VLCDR2 amino acid sequence ofSEQ ID NO:67-69, and a VLCDR3 amino acid sequence of SEQ ID NO:63-66.

Embodiment 13

An isolated nucleic acid encoding an antigen binding protein accordingto Embodiment 1.

Embodiment 14

An expression vector comprising the nucleic acid according to Embodiment13.

Embodiment 15

A prokaryotic or eukaryotic host cell comprising a vector of Embodiment14.

Embodiment 16

A method for the production of a recombinant protein comprising thesteps of expressing a nucleic acid according to Embodiment 13 in aprokaryotic or eukaryotic host cell and recovering said protein fromsaid cell or the cell culture supernatant.

Embodiment 17

A method for treatment of a subject suffering from cancer or from aninflammatory disease, comprising the step of administering to thesubject a pharmaceutical composition comprising an effective amount ofthe antigen binding protein of Embodiment 1.

Embodiment 18

The method of Embodiment 17, wherein the cancer is a solid cancer.

Embodiment 19

The method of Embodiment 17, wherein the cancer is a hematologicalcancer.

Embodiment 20

A method for modulating immune system function in a human subject inneed thereof, comprising the step of contacting a population of T cellsof the human subject with a pharmaceutical composition comprising aneffective amount of the antigen binding protein of Embodiment 1, underconditions such that the immune system is modulated.

Embodiment 21

A method for inducing or enhancing an immune response in a subject,comprising the step of administering to the subject a pharmaceuticalcomposition comprising an antigen binding protein or a bispecificantibody or a complexing antigen binding protein, of any of thepreceding embodiments, wherein the immune response is generated againsta tumor antigen.

Embodiment 22

The method of Embodiment 21, wherein the antigen binding protein,bispecific antibody or the complexing antigen binding protein isadministered in an amount sufficient to achieve one or more of thefollowing in the subject: a) reduce regulatory T cells suppression ofactivity of effector T cells; b) decrease levels of regulatory T cells;c) activation of effector T cells; d) induce or enhance effector T cellproliferation; e) inhibit tumor growth; and f) induce tumor regression.

Embodiment 23

The method of Embodiment 22, wherein the method further comprises one ormore of the following a) administering chemotherapy; b) administeringradiation therapy; or c) administering one or more additionaltherapeutic agents.

Embodiment 24

The method of Embodiment 23, wherein the additional therapeutic agentcomprises an immunostimulatory agent.

Embodiment 25

The method of Embodiment 24, wherein the immunostimulatory agentcomprises an antagonist to an inhibitory receptor of an immune cell.

Embodiment 26

The method of Embodiment 25, wherein the inhibitory receptor is CTLA-4,PD-1, PD-L1, PD-L2, LAG-3, Tim3, neuritin, BTLA, CECAM-1, CECAM-5,VISTA, LAIR1, CD160, 2B4, TGF-R, or a KIR.

Embodiment 27

The method of Embodiment 24, wherein the immunostimulatory agentcomprises an agonist of co-stimulatory receptor of an immune cell.

Embodiment 28

The method of Embodiment 27, wherein the co-stimulatory receptor isOX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB(CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7,NKp80, CD160, B7-H3 or CD83 ligand.

Embodiment 29

The method of Embodiment 24, wherein the immunostimulatory agentcomprises a cytokine.

Embodiment 30

The method of Embodiment 29, wherein the cytokine is IL-2, IL-5, IL-7,IL-12, IL-15 or IL-21.

Embodiment 31

The method of Embodiment 24, wherein the immunostimulatory agentcomprises an oncolytic virus.

Embodiment 32

The method of Embodiment 31, wherein the oncolytic virus is a Herpessimplex virus, a Vesicular stomatitis virus, an adenovirus, a Newcastledisease virus, a vaccinia virus, or a maraba virus.

Embodiment 33

The method of Embodiment 24, wherein the immunostimulatory agentcomprises a chimeric antigen engineered T cell.

Embodiment 34

The method of Embodiment 24, wherein the immunostimulatory agentcomprises a bi- or multispecific T cell directed antibody.

Embodiment 35

The method of Embodiment 23, wherein the additional therapeutic agentcomprises an anti-TGF-beta antibody or a TGFβ receptor trap.

Embodiment 36

The method of any one of Embodiments 20-35, wherein administration ofthe pharmaceutical composition results in induction or enhancement ofproliferation of a T-effector cell, or modulation of I-κB and/or NF-κBin the T cell, or modulation of TIGIT activity in the T cell, or T cellreceptor induced signaling in a T-effector cell, or a combinationthereof.

Embodiment 37

A method of screening for test compounds comprising an antigen bindingprotein of Embodiment 1 that are capable of inhibiting the interactionof a TIGIT ligand with TIGIT, comprising the steps of: contacting asample containing a TIGIT ligand and TIGIT with the compound; anddetermining whether the interaction of a TIGIT ligand with TIGIT in thesample is decreased relative to the interaction of a TIGIT ligand withTIGIT in a sample not contacted with the compound, whereby a decrease inthe interaction of a TIGIT ligand with TIGIT in the sample contactedwith the compound identifies the compound as one that inhibits theinteraction of a TIGIT ligand with TIGIT.

Embodiment 38

The antigen binding protein of Embodiment 1, that is capable ofinhibiting phosphorylation of the ITIM domain of the TIGIT polypeptide.

Embodiment 1A

An isolated antigen-binding protein (ABP) that specifically binds toTIGIT, wherein the antibody: (a) competes for binding to TIGIT with anantibody selected from MAB1, MAB2, MAB3, MAB4, MAB5, MAB6, MAB7, MAB8,MAB9, MAB10, MAB11, MAB12, MAB13, MAB14, MAB15, MAB16, MAB17, MAB18,MAB19, MAB20, or MAB21, each as provided in Table 5 of this disclosure;(b) inhibits binding of CD155 to TIGIT; (c) inhibits binding of CD112 toTIGIT; (d) inhibits association of CD226 with TIGIT; (e) activates aneffector T cell or an NK cell; (f) decreases the number of regulatory Tcells in a tissue or in circulation; (g) inhibits the suppression of aneffector T cell by a regulatory T cell; (h) does not bind specificallyto any of PVRL1, PVRL2, PVRL3, or PVRL4; or (i) is capable of anycombination of (a)-(h).

Embodiment 2A

The ABP of Embodiment 1A, wherein the ABP comprises a CDR-H3 of a V_(H)region selected from SEQ ID NOs: 4-24, or a CDR-H3 having at least about80% identity to a CDR-H3 of a V_(H) region selected from SEQ ID NOs:4-24.

Embodiment 3A

The ABP of Embodiment 2A, wherein the CDR-H3 is identified according tothe Kabat, Chothia, or IMGT numbering schemes.

Embodiment 4A

The ABP of any of Embodiments 2A-3A, wherein the CDR-H3 is selected fromSEQ ID NOs: 29-35.

Embodiment 5A

The ABP of any of Embodiments 1A-4A, wherein the ABP comprises a CDR-H2of a V_(H) region selected from SEQ ID NOs: 4-24, or a CDR-H2 having atleast about 80% identity to a CDR-H2 of a V_(H) region selected from SEQID NOs: 4-24

Embodiment 6A

The ABP of Embodiment 5A, wherein the CDR-H2 is identified according tothe Kabat, Chothia, or IMGT numbering schemes.

Embodiment 7A

The ABP of any of Embodiments 5A-6A, wherein the CDR-H2 is selected fromSEQ ID NOs: 36-47.

Embodiment 8A

The ABP of any of Embodiments 1A-7A, wherein the ABP comprises a CDR-H1of a V_(H) region selected from SEQ ID NOs: 4-24, or a CDR-H1 having atleast about 80% identity to a CDR-H1 of a V_(H) region selected from SEQID NOs: 4-24

Embodiment 9A

The ABP of Embodiment 8A, wherein the CDR-H1 is identified according tothe Kabat, Chothia, Kabat plus Chothia, or IMGT numbering schemes.

Embodiment 10A

The ABP of any of Embodiments 8A-9A, wherein the CDR-H1 is selected fromSEQ ID NOs: 48-54 and 58-62.

Embodiment 11A

The ABP of any of Embodiments 1A-10A, wherein the ABP comprises a CDR-L3of a V_(L) region selected from SEQ ID NOs: 25-28, or a CDR-L3 having atleast about 80% identity to a CDR-L3 of a V_(L) region selected from SEQID NOs: 25-28.

Embodiment 12A

The ABP of Embodiment 11A, wherein the CDR-L3 is identified according tothe Kabat, Chothia, or IMGT numbering schemes.

Embodiment 13A

The ABP of any of Embodiments 11A-12A, wherein the CDR-L3 is selectedfrom SEQ ID NOs: 63-66.

Embodiment 14A

The ABP of any of Embodiments 1A-13A, wherein the ABP comprises a CDR-L2of a V_(L) region selected from SEQ ID NOs: 25-28, or a CDR-L2 having atleast about 80% identity to a CDR-L2 of a V_(L) region selected from SEQID NOs: 25-28.

Embodiment 15A

The ABP of Embodiment 14A, wherein the CDR-L2 is identified according tothe Kabat, Chothia, or IMGT numbering schemes.

Embodiment 16A

The ABP of any of Embodiments 14A-15A, wherein the CDR-L2 is selectedfrom SEQ ID NOs: 67-69.

Embodiment 17A

The ABP of any of Embodiments 1A-16A, wherein the ABP comprises a CDR-L1of a V_(L) region selected from SEQ ID NOs: 25-28, or a CDR-L1 having atleast about 80% identity to a CDR-L1 of a V_(L) region selected from SEQID NOs: 25-28.

Embodiment 18A

The ABP of Embodiment 17A, wherein the CDR-L1 is identified according tothe Kabat, Chothia, or IMGT numbering schemes.

Embodiment 19A

The ABP of any of Embodiments 17A-18A, wherein the CDR-L1 is selectedfrom SEQ ID NOs: 70-72.

Embodiment 20A

The ABP of any of Embodiments 1A-19A, wherein the ABP comprises a V_(H)region selected from SEQ ID NOs: 4-24.

Embodiment 21A

The ABP of any of Embodiments 1A-20A, wherein the ABP comprises a V_(L)region selected from SEQ ID NOs: 25-28.

Embodiment 22A

The ABP of any of Embodiments 1A-21A, wherein the TIGIT is selected fromhTIGIT (SEQ ID NO: 1), cTIGIT (SEQ ID NO: 2), and mTIGIT (SEQ ID NOs: 3or 138).

Embodiment 23A

The ABP of any of Embodiments 1A-22A, wherein the ABP comprises anantibody.

Embodiment 24A

The ABP of Embodiment 23A, wherein the antibody comprises a V_(H) andV_(L) paired as provided for an antibody selected from MAB1, MAB2, MAB3,MAB4, MAB5, MAB6, MAB7, MAB8, MAB9, MAB10, MAB11, MAB12, MAB13, MAB14,MAB15, MAB16, MAB17, MAB18, MAB19, MAB20, or MAB21, each as provided inTable 5 of this disclosure.

Embodiment 25A

The ABP of Embodiment 24A, wherein the ABP is an antibody selected fromMAB1, MAB2, MAB3, MAB4, MAB5, MAB6, MAB7, MAB8, MAB9, MAB10, MAB11,MAB12, MAB13, MAB14, MAB15, MAB16, MAB17, MAB18, MAB19, MAB20, or MAB21,each as provided in Table 5 of this disclosure.

Embodiment 26A

The ABP of any of Embodiments 23A-25A, wherein the antibody is amonoclonal antibody.

Embodiment 27A

The ABP of any of Embodiments 23A-26A, wherein the antibody is achimeric, humanized, or human antibody

Embodiment 28A

The ABP of any of Embodiments 1A-27A, wherein the ABP is multispecific.

Embodiment 29A

The ABP of any of Embodiments 1A-28A, wherein the ABP comprises anantibody fragment.

Embodiment 30A

The ABP of any of Embodiments 1A-29A, wherein the ABP comprises analternative scaffold.

Embodiment 31A

The ABP of any of Embodiments 1A-30A, wherein the ABP comprises animmunoglobulin constant region.

Embodiment 32A

The ABP of any of Embodiments 1A-31A, wherein the ABP comprises anantibody selected from an IgA, an IgD, an IgE, an IgG, or an IgM.

Embodiment 33A

The ABP of Embodiment 32A, wherein the ABP comprises an IgG selectedfrom an IgG4, an IgG1, an IgG2, or an IgG3.

Embodiment 34A

The ABP of any of Embodiments 1A-33A, wherein the ABP binds hTIGIT (SEQID NO: 1) with an affinity of less than about 20 nM.

Embodiment 35A

The ABP of any of Embodiments 1A-34A, wherein the ABP binds cTIGIT (SEQID NO: 2) with an affinity of less than about 200 nM.

Embodiment 36A

The ABP of any of Embodiments 1A-35A, wherein the ABP binds mTIGIT (SEQID NO: 3 or 138) with an affinity of less than about 200 nM.

Embodiment 37A

An isolated polynucleotide encoding an ABP of any of Embodiments 1A-36A,a V_(H) or V_(L) thereof, or an antigen-binding portion thereof.

Embodiment 38A

A vector comprising the polynucleotide of Embodiment 37A.

Embodiment 39A

A host cell comprising the vector of Embodiment 38A.

Embodiment 40A

A method of producing an ABP of any of Embodiments 1A-36A, comprisingexpressing the ABP in the host cell of Embodiment 39A and isolating theexpressed ABP.

Embodiment 41A

A pharmaceutical composition comprising an ABP of any of Embodiments1A-36A.

Embodiment 42A

The pharmaceutical composition of Embodiment 41A, wherein the amount ofthe ABP in the pharmaceutical composition is sufficient to (a) increaseeffector T cell activity; (b) increase cytolytic T cell activity; (c)increase NK cell activity; (d) inhibit TIGIT-mediated signaling; (e)inhibit or block the binding of CD155 and or CD112 to TIGIT; or (f) anycombination of (a)-(e), in a subject.

Embodiment 43A

The pharmaceutical composition of any of Embodiments 41A-42A, furthercomprising an antibody that antagonizes PD-1.

Embodiment 44A

A method of treating or preventing a disease or condition in a subjectin need thereof, comprising administering to the subject an effectiveamount of an ABP of any of Embodiments 1A-36A or a pharmaceuticalcomposition of any of Embodiments 41A-43A.

Embodiment 45A

The method of Embodiment 44A, wherein the disease or condition is acancer or viral infection.

Embodiment 46A

A method of modulating an immune response in a subject in need thereof,comprising administering to the subject an effective amount of an ABP ofany of Embodiments 1A-36A or a pharmaceutical composition of any ofEmbodiments 41A-43A.

Embodiment 47A

The method of any of Embodiments 44A-46A, further comprisingadministering one or more additional therapeutic agents to the subject.

Embodiment 48A

The method of Embodiment 47A, wherein the additional therapeutic agentis selected from a PD-1 antagonist antibody, a chemotherapy, animmunostimulatory agent, and radiation.

Embodiment 49A

The method of Embodiments 47A, wherein the additional therapeutic agentis an immunostimulatory agent that blocks signaling of an inhibitoryreceptor of an immune cell or a ligand thereof.

Embodiment 50A

The method of Embodiment 49A, wherein the inhibitory receptor or ligandthereof is selected from CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, Tim3,neuritin, BTLA, CECAM-1, CECAM-5, VISTA, LAIR1, CD160, 2B4, TGF-R, KIR,and combinations thereof.

Embodiment 51A

The method of Embodiment 48A, wherein the additional therapeutic agentis an immunostimulatory agent that is an agonist to a stimulatoryreceptor of an immune cell.

Embodiment 52A

The method of Embodiment 51A, wherein the stimulatory receptor of animmune cell is selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40,BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83ligand, and combinations thereof.

Embodiment 53A

The method of Embodiment 48A, wherein the additional therapeutic agentis an immunostimulatory agent that is a cytokine.

Embodiment 54A

The method of Embodiment 53A, wherein the cytokine is selected fromIL-2, IL-5, IL-7, IL-12, IL-15, IL-21, and combinations thereof.

Embodiment 55A

The method of Embodiment 48A, wherein the additional therapeutic agentis an immunostimulatory agent that is an oncolytic virus.

Embodiment 56A

The method of Embodiment 55A, wherein the oncolytic virus is selectedfrom herpes simplex virus, vesicular stomatitis virus, adenovirus,Newcastle disease virus, vaccinia virus, a maraba virus, andcombinations thereof.

Embodiment 57A

The method of Embodiment 48A, wherein the immunostimulatory agentcomprises a T cell expressing a chimeric antigen receptor.

Embodiment 58A

The method of Embodiment 48A, wherein the immunostimulatory agentcomprises a bi- or multi-specific T cell directed antibody.

Embodiment 59A

The method of Embodiment 48A, wherein the immunostimulatory agentcomprises an anti-TGF-β antibody, a TGF-β trap, or a combinationthereof.

Embodiment 60A

The method of Embodiment 48A, wherein the immunostimulatory agentcomprises a vaccine to a cancer-associated antigen.

Embodiment 61A

A method of screening for ABPs capable of inhibiting the interaction ofa ligand of TIGIT with TIGIT, comprising (a) contacting a samplecomprising a ligand of TIGIT and TIGIT with an ABP of any of Embodiments1A-36A, and (b) determining if the binding of the ligand of TIGIT toTIGIT is decreased in the presence of the ABP, in comparison to thebinding of the ligand of TIGIT to TIGIT in the absence of the ABP.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided herein.

Example 1: Selection of TIGIT Antigen-Binding Proteins

TIGIT ABPs were selected from a synthetic library of human antibodiesexpressed and displayed on the surface of yeast cells in IgG format, asgenerally described, e.g., in WO2009036379; WO2010105256; WO2012009568;and Xu et al., Protein Eng. Des. Sel., 2013, 26:663-670 (eachincorporated by reference in its entirety), and more specifically asprovided below. The sequences and characteristics of the ABPs isolatedfrom the recombinant library are provided in Table 5.

Eight naïve human synthetic yeast libraries each of ˜10⁹ diversity werepropagated as described in WO2009036379; WO2010105256; WO2012009568; andXu et al., Protein Eng. Des. Sel., 2013, 26:663-670; each incorporatedby reference in its entirety. For the first two rounds of selection, amagnetic bead sorting technique utilizing the Miltenyi MACS® system wasperformed, as described in Siegel et al., J. Immunol. Meth., 2004,286:141-153. Briefly, yeast cells (˜10¹⁰ cells/library) were incubatedwith biotinylated TIGIT-Fc antigen in FACS wash buffer(phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA)). Afterwashing once with 50 ml ice-cold wash buffer, the cell pellet wasresuspended in 40 mL wash buffer, and 500 μl streptavidin MicroBeads™(Miltenyi Biotec) were added to the yeast and incubated for 15 min at 4°C. Next, the yeast were pelleted, resuspended in 5 mL wash buffer, andloaded onto a Miltenyi LS column. After the 5 mL was loaded, the columnwas washed 3 times with 3 ml FACS wash buffer. The column was thenremoved from the magnetic field, and the yeast were eluted with 5 mL ofgrowth media and then grown overnight. The following rounds of sortingwere performed using flow cytometry. Approximately 1×10⁸ yeast werepelleted, washed three times with wash buffer, and incubated withdecreasing concentrations of biotinylated TIGIT-Fc fusion antigen (100to 1 nM) under equilibrium conditions at room temperature. Yeast werethen washed twice and stained with LC-FITC (diluted 1:100) and eitherSA-633 (diluted 1:500) or EA-PE (diluted 1:50) secondary reagents for 15min at 4° C. After washing twice with ice-cold wash buffer, the cellpellets were resuspended in 0.4 mL wash buffer and transferred tostrainer-capped sort tubes. Sorting was performed using a FACS ARIAsorter (BD Biosciences) and sort gates were assigned to select forspecific binders relative to a background control. Subsequent rounds ofselection were employed in order to reduce the number of non-specificbinders utilizing soluble membrane proteins from CHO cells (seeWO2014179363 and Xu et al., Protein Eng. Des. Sel., 2013, 26:663-670,each incorporated by reference in its entirety), and identify binderswith improved affinity to TIGIT using the TIGIT-Fc antigen. After thefinal round of sorting, yeast were plated and individual colonies werepicked for characterization and for nomination of clones for affinitymaturation.

Example 2: Affinity Maturation

Optimization of naïve clones was carried out utilizing three maturationstrategies: light chain diversification; diversification of CDR-H1 andCDR-H2; and performing VH mutagenesis.

Light Chain Diversification:

Heavy chain plasmids were extracted from naïve outputs (described above)and transformed into a light chain library with a diversity of 1×10⁶.Selections were performed as described above with one round of MACSsorting and two rounds of FACS sorting using 10 nM or 1 nM biotinylatedTIGIT-Fc antigen for respective rounds.

CDR-H1 and CDR-H2 Selection:

The CDR-H3s from clones selected from the light chain diversificationprocedure were recombined into a premade library with CDR-H1 and CDR-H2variants of a diversity of 1×10⁸ and selections were performed usingmonomeric HIS-TIGIT antigen. Affinity pressures were applied by usingdecreasing concentrations of biotinylated HIS-TIGIT antigen (100 to 1nM) under equilibrium conditions at room temperature.

V_(H)mut Selection:

Clones obtained from the CDR-H1 and CDR-H2 selection procedure weresubject to additional rounds of affinity maturation via error pronePCR-based mutagenesis of the heavy chain. Selections were performedusing HIS-TIGIT as antigen generally as described above but with theaddition of employing FACS sorting for all selection rounds.

Example 3: Antibody Production and Purification

In order to produce sufficient amounts of selected antibodies forfurther characterization, the yeast clones were grown to saturation andthen induced for 48 h at 30° C. with shaking. After induction, yeastcells were pelleted and the supernatants were harvested forpurification. IgGs were purified using a Protein A column and elutedwith acetic acid, pH 2.0. Fab fragments were generated by papaindigestion and purified over KappaSelect® (GE Healthcare LifeSciences).

Antibodies were also produced by transient transfection of Expi293 cellsaccording to the manufacturer's protocol (Thermo Fisher), transienttransfection of CHO cells, or stable expression of CHO cells. Antibodieswere purified by Protein A chromatography.

TABLE 5 Sequences and germlines (GL) of TIGIT ABPs. VH CDR- CDR- CDR- VLCDR- CDR- CDR- Ab GL H1¹ H2² H3³ VH Protein GL L1⁴ L2⁵ L3⁶ VL ProteinMAB1- VH4- GSITSS SIYYSG ARDAN QLQLQESGPGLVKPSETLSLT VK3- RASQS DASNRQQHFN EIVLTQSPATLSLSP IgG4 39 SYYWG ATFYN YYGSA CTVSGGSITSSSYYWGWIRQP 11VSSYL AT LPT GERATLSCRASQSV (SEQ ID PSLKS WAFDP PGKGLEWIGSIYYSGATFYNP A(SEQ (SEQ ID SSYLAWYQQKPGQ NO: 48) (SEQ ID (SEQ IDSLKSRVTISVDTSKNQFSLKLS (SEQ ID NO: 63) APRLLIYDASNRAT NO: 36) NO: 29)SVTAADTAVYYCARDANYYG ID NO: NO: GIPARFSGSGSGTDF SAWAFDPWGQGTLVTVSS 70)67) TLTISSLEPEDFAVY (SEQ ID NO: 4) YCQQHFNLPTFGGG TKVEIK (SEQ ID NO: 25)MAB2- VH4- GSISSS SIYYSG ARDAN QLQLQESGPGLVKPSETLSLT VK3- RASQS DASNRQQHFN EIVLTQSPATLSLSP IgG4 39 KYYWG STFYN YYGSA CTVSGGSISSSKYYWGWIRQP 11VSSYL AT LPT GERATLSCRASQSV (SEQ ID PSLKS WAFDP PGKGLEWIGSIYYSGSTFYNPS A(SEQ (SEQ ID SSYLAWYQQKPGQ NO: 49) (SEQ ID (SEQ IDLKSRVTISVDTSKNQFSLKLSS (SEQ ID NO: 63) APRLLIYDASNRAT NO: 37) NO: 29)VTAADTAVYYCARDANYYGS ID NO: NO: GIPARFSGSGSGTDF AWAFDPWGQGTLVTVSS 70)67) TLTISSLEPEDFAVY (SEQ ID NO: 5) YCQQHFNLPTFGGG TKVEIK (SEQ ID NO: 25)MAB3- VH4- GSISST SIYYSG ARDAN QLQLQESGPGLVKPSETLSLT VK3- RASQS DASNRQQHFN EIVLTQSPATLSLSP IgG4 39 SHYWG STFYN YYGSA CTVSGGSISSTSHYWGWIRQP 11VSSYL AT LPT GERATLSCRASQSV (SEQ ID PSLKS WAFDP PGKGLEWIGSIYYSGSTFYNPS A(SEQ (SEQ ID SSYLAWYQQKPGQ NO: 50) (SEQ ID (SEQ IDLKSRVTISVDTSKNQFSLKLSS (SEQ ID NO: 63) APRLLIYDASNRAT NO: 37) NO: 29)VTAADTAVYYCARDANYYGS ID NO: NO: GIPARFSGSGSGTDF AWAFDPWGQGTLVTVSS 70)67) TLTISSLEPEDFAVY (SEQ ID NO: 6) YCQQHFNLPTFGGG TKVEIK (SEQ ID NO: 25)MAB4- VH4- GSISST SIYYSG ARDAN QLQLQESGPGLVKPSETLSLT VK3- RASQS DASNRQQHFN EIVLTQSPATLSLSP IgG4 39 SHYWG STFYN YYGGA CTVSGGSISSTSHYWGWIRQP 11VSSYL AT LPT GERATLSCRASQSV (SEQ ID PSLKS WAFDP PGKGLEWIGSIYYSGSTFYNPS A(SEQ (SEQ ID SSYLAWYQQKPGQ NO: 50) (SEQ ID (SEQ IDLKSRVTISVDTSKNQFSLKLSS (SEQ ID NO: 63) APRLLIYDASNRAT NO: 37) NO: 30)VTAADTAVYYCARDANYYG ID NO: NO: GIPARFSGSGSGTDF GAWAFDPWGQGTLVTVSS 70)67) TLTISSLEPEDFAVY (SEQ ID NO: 7) YCQQHFNLPTFGGG TKVEIK (SEQ ID NO: 25)MAB5- VH4- GSISST SIYYSG ARDAN QLQLQESGPGLVKPSETLSLT VK3- RASQS DASNRQQHFN EIVLTQSPATLSLSP IgG4 39 SHYWG STFYN YYGSA CTVSGGSISSTSHYWGWIRQP 11VSSYL AT LPT GERATLSCRASQSV (SEQ ID PSLKG WAFDP PGKGLEWIGSIYYSGSTFYNPS A(SEQ (SEQ ID SSYLAWYQQKPGQ NO: 50) (SEQ ID (SEQ IDLKGRVTISVDTSKNQFSLKLSS (SEQ ID NO: 63) APRLLIYDASNRAT NO: 38) NO: 29)VTAADTAVYYCARDANYYGS ID NO: NO: GIPARFSGSGSGTDF AWAFDPWGQGTLVTVSS 70)67) TLTISSLEPEDFAVY (SEQ ID NO: 8) YCQQHFNLPTFGGG TKVEIK (SEQ ID NO: 25)MAB6- VH4- GSIESG SIYYSG ARDGV QLQLQESGPGLVKPSETLSLT VK3- RASQS GASSRQQHTV EIVLTQSPGTLSLSP IgG4 39 SYYWG GTYYN LTLNK CTVSGGSIESGSYYWGWIRQP 20VSSSY AT RPPLT GERATLSCRASQSV (SEQ ID PSLKS RSFDI PGKGLEWIGSIYYSGGTYYNPLA (SEQ (SEQ ID SSSYLAWYQQKPGQ NO: 51) (SEQ ID (SEQ IDSLKSRVTISVDTSKNQFSLKLS (SEQ ID NO: 64) APRLLIYGASSRATG NO: 39) NO: 31)SVTAADTAVYYCARDGVLTL ID NO: NO: IPDRFSGSGSGTDFT NKRSFDIWGQGTMVTVSS 71)68) LTISRLEPEDFAVYY (SEQ ID NO: 9) CQQHTVRPPLTFGG GTKVEIK(SEQ ID NO: 26) MAB7- VH4- GSIESG SIYYSG ARDGV QVQLQESGPGLVKPSQTLSLTVK3- RASQS GASSR QQHTV EIVLTQSPGTLSLSP IgG4 31 VYYWG STYYN LTLNKCTVSGGSIESGVYYWGWIRQP 20 VSSSY AT RPPLT GERATLSCRASQSV (SEQ ID PSLKSRSFDI PGKGLEWIGSIYYSGSTYYNP LA (SEQ (SEQ ID SSSYLAWYQQKPGQ NO: 52)(SEQ ID (SEQ ID SLKSRVTISVDTSKNQFSLKLS (SEQ ID NO: 64) APRLLIYGASSRATGNO: 40) NO: 31) SVTAADTAVYYCARDGVLTL ID NO: NO: IPDRFSGSGSGTDFTNKRSFDIWGQGTMVTVSS 71) 68) LTISRLEPEDFAVYY (SEQ ID NO: 10)CQQHTVRPPLTFGG GTKVEIK (SEQ ID NO: 26) MAB8- VH4- GSIASG SIYYSG ARDGVQLQLQESGPGLVKPSETLSLT VK3- RASQS GASSR QQHTV EIVLTQSPGTLSLSP IgG4 39SYYWG QTYYN LTLNK CTVSGGSIASGSYYWGWIRQP 20 VSSSY AT RPPLT GERATLSCRASQSV(SEQ ID PSLKS RSFDI PGKGLEWIGSIYYSGQTYYNP LA (SEQ (SEQ ID SSSYLAWYQQKPGQNO: 53) (SEQ ID (SEQ ID SLKSRVTISVDTSKNQFSLKLS (SEQ ID NO: 64)APRLLIYGASSRATG NO: 41) NO: 31) SVTAADTAVYYCARDGVLTL ID NO: NO:IPDRFSGSGSGTDFT NKRSFDIWGQGTMVTVSS 71) 68) LTISRLEPEDFAVYY(SEQ ID NO: 11) CQQHTVRPPLTFGG GTKVEIK (SEQ ID NO: 26) MAB9- VH4- GSIESGSIYYSG ARDGV QVQLQESGPGLVKPSQTLSLT VK3- RASQS GASSR QQHTVEIVLTQSPGTLSLSP IgG4 31 LYYWG STYYN LTLNK CTVSGGSIESGLYYWGWIRQP 20 VSSSYAT RPPLT GERATLSCRASQSV (SEQ ID PSLKS RSFDI PGKGLEWIGSIYYSGSTYYNP LA(SEQ (SEQ ID SSSYLAWYQQKPGQ NO: 54) (SEQ ID (SEQ IDSLKSRVTISVDTSKNQFSLKLS (SEQ ID NO: 64) APRLLIYGASSRATG NO: 40) NO: 31)SVTAADTAVYYCARDGVLTL ID NO: NO: IPDRFSGSGSGTDFT NKRSFDIWGQGTMVTVSS 71)68) LTISRLEPEDFAVYY (SEQ ID NO: 12) CQQHTVRPPLTFGG GTKVEIK(SEQ ID NO: 26) MAB10- VH4- GSIESG SIYYSG ARDGV QVQLQESGPGLVKPSQTLSLTVK3- RASQS GASSR QQHTV EIVLTQSPGTLSLSP IgG4 31 LYYWG STYYN LALNKCTVSGGSIESGLYYWGWIRQP 20 VSSSY AT RPPLT GERATLSCRASQSV (SEQ ID PSLKSRSFDI PGKGLEWIGSIYYSGSTYYNP LA (SEQ (SEQ ID SSSYLAWYQQKPGQ NO: 54)(SEQ ID (SEQ ID SLKSRATISVDTSKNQFSLKLS (SEQ ID NO: 64) APRLLIYGASSRATGNO: 40) NO: 32) SVTAADTAVYYCARDGVLAL ID NO: NO: IPDRFSGSGSGTDFTNKRSFDIWGQGTMVTVSS 71) 68) LTISRLEPEDFAVYY (SEQ ID NO: 13)CQQHTVRPPLTFGG GTKVEIK (SEQ ID NO: 26) MAB11- VH4- GSIESG SIYYSG ARDGVQVQLQESGPGLVKPSQTLSLT VK3- RASQS GASSR QQHTV EIVLTQSPGTLSLSP IgG4 31LYYWG STYYN LALNK CTVSGGSIESGLYYWGWIRQP 20 VSSSY AT RPPLT GERATLSCRASQSV(SEQ ID PSLKS RSFDI PGKGLEWIGSIYYSGSTYYNP LA (SEQ (SEQ ID SSSYLAWYQQKPGQNO: 54) (SEQ ID (SEQ ID SLKSRVTISVDTSKNQFSLKLS (SEQ ID NO: 64)APRLLIYGASSRATG NO: 40) NO: 32) SVTAADTAVYYCARDGVLAL ID NO: NO:IPDRFSGSGSGTDFT NKRSFDIVVGQGTMVTVSS 71) 68) LTISRLEPEDFAVYY(SEQ ID NO: 14) CQQHTVRPPLTFGG GTKVEIK (SEQ ID NO: 26) MAB12- VH4-GSIESG SIYYSG ARDGV QVQLQESGPGLVKPSQTLSLT VK3- RASQS GASSR QQHTVEIVLTQSPGTLSLSP IgG4 31 LYYWG STYYN LALNK CTASGGSIESGLYYWGWIRQP 20 VSSSYAT RPPLT GERATLSCRASQSV (SEQ ID PSLKS RSFDI PGKGLEWIGSIYYSGSTYYNP LA(SEQ (SEQ ID SSSYLAWYQQKPGQ NO: 54) (SEQ ID (SEQ IDSLKSRVTISVDTSKNQFSLKLS (SEQ ID NO: 64) APRLLIYGASSRATG NO: 40) NO: 32)SVTAADTAVYYCARDGVLAL ID NO: NO: IPDRFSGSGSGTDFT NKRSFDIWGQGTMVTVSS 71)68) LTISRLEPEDFAVYY (SEQ ID NO: 15) CQQHTVRPPLTFGG GTKVEIK(SEQ ID NO: 26) MAB13- VH1- YTFGN IINPSL ARGGR QVQLVQSGAEVKKPGASVKV VK3-RASQS GASTR QQYVV EIVMTQSPATLSVSP IgG4 46 YYMH GLTSY TTWIGSCKASGYTFGNYYMHWVRQ 15 VSSNL AT WPPLT GERATLSCRASQSV (SEQ ID AQKFQ AFDIAPGQGLEWMGINPSLGLTSY A (SEQ (SEQ ID SSNLAWYQQKPGQ NO: 58) G (SEQ IDAQKFQGRVTMTRDTSTSTVY (SEQ ID NO: 65) APRLLIYGASTRATG (SEQ ID NO: 33)MELSSLRSEDTAVYYCARGG ID NO: NO: IPARFSGSGSGTEFT NO: 42)RTTWIGAFDIWGQGTMVTVS 72) 69) LTISSLQSEDFAVYY S CQQYVVWPPLTFGG(SEQ ID NO: 16) GTKVEIK (SEQ ID NO: 27) MAB14- VH1- YTFPA IINPSL ARGGRQVQLVQSGAEVKKPGASVKV VK3- RASQS GASTR QQYVV EIVMTQSPATLSVSP IgG4 46 YYMHGLTSY TTWIG SCKASGYTFPAYYMHWVRQA 15 VSSNL AT WPPLT GERATLSCRASQSV(SEQ ID AQKFQ AFDI PGQGLEWMGIINPSLGLTSYA A (SEQ (SEQ ID SSNLAWYQQKPGQNO: 59) G (SEQ ID QKFQGRVTMTRDTSTSTVYM (SEQ ID NO: 65) APRLLIYGASTRATG(SEQ ID NO: 33) ELSSLRSEDTAVYYCARGGRT ID NO: NO: IPARFSGSGSGTEFT NO: 42)TWIGAFDIWGQGTMVTVSS 72) 69) LTISSLQSEDFAVYY (SEQ ID NO: 17)CQQYVVWPPLTFGG GTKVEIK (SEQ ID NO: 27) MAB15- VH1- YTFRE IINPSIG ARGGRQVQLVQSGAEVKKPGASVKV VK3- RASQS GASTR QQYVV EIVMTQSPATLSVSP IgG4 46 YYMHLTSYA TTWIG SCKASGYTFREYYMHWVRQA 15 VSSNL AT WPPLT GERATLSCRASQSV(SEQ ID RKFQG AFDI PGQGLEWMGIINPSIGLTSYAR A (SEQ (SEQ ID SSNLAWYQQKPGQNO: 60) (SEQ ID (SEQ ID KFQGRVTMTRDTSTSTVYME (SEQ ID NO: 65)APRLLIYGASTRATG NO: 43) NO: 33) LSSLRSEDTAVYYCARGGRTT ID NO: NO:IPARFSGSGSGTEFT WIGAFDIWGQGTMVTVSS 72) 69) LTISSLQSEDFAVYY(SEQ ID NO: 18) CQQYVVWPPLTFGG GTKVEIK (SEQ ID NO: 27) MAB16- VH1- YTFREIINPSIG ARGGR QVQLVQSGAEVKKPGASVKV VK3- RASQS GASTR QQYVVEIVMTQSPATLSVSP IgG4 46 YYMH LTSYA TTWIG SCKASGYTFREYYMHWVRQA 15 VSSNLAT WPPLT GERATLSCRASQSV (SEQ ID RKFQG ALDI PGQGLEWMGIINPSIGLTSYAR A (SEQ(SEQ ID SSNLAWYQQKPGQ NO: 60) (SEQ ID (SEQ ID KFQGRVTMTRDTSTSTVYME (SEQID NO: 65) APRLLIYGASTRATG NO: 43) NO: 34) LSSLRSEDTAVYYCARGGRTT ID NO:NO: IPARFSGSGSGTEFT WIGALDIWGQGTMVTVSS 72) 69) LTISSLQSEDFAVYY(SEQ ID NO: 19) CQQYVVWPPLTFGG GTKVEIK (SEQ ID NO: 27) MAB17- VH1- YTFPAIINPSL ARGGR QVQLVQSGAEVKKPGASVKV VK3- RASQS GASTR QQYVV EIVMTQSPATLSVSPIgG4 46 YY1F1 GLTSY TTWIG SCKASGYTFPAYYIHWVRQAP 15 VSSNL AT WPPLTGERATLSCRASQSV (SEQ ID ARKFQ ALDI GQGLEWMGIINPSLGLTSYAR A (SEQ (SEQ IDSSNLAWYQQKPGQ NO: 61) G (SEQ ID KFQGRVTMTRDTSTSTVYME (SEQ ID NO: 65)APRLLIYGASTRATG (SEQ ID NO: 34) LSSLRSEDTAVYYCARGGRTT ID NO: NO:IPARFSGSGSGTEFT NO: 44) WIGALDIVVGQGTMVTVSS 72) 69) LTISSLQSEDFAVYY(SEQ ID NO: 20) CQQYVVWPPLTFGG GTKVEIK (SEQ ID NO: 27) MAB18- VH1- YTFPAIINPSL ARGGR QVQLVQSGAEVKKPGASVKV VK3- RASQS GASTR QQYVV EIVMTQSPATLSVSPIgG4 46 YYMH GLTSY TTWIG SCKASGYTFPAYYMHWVRQA 15 VSSNL AT WPPLTGERATLSCRASQSV (SEQ ID ARKFQ AFDI PGQGLEWMGIINPSLGLTSYA A (SEQ (SEQ IDSSNLAWYQQKPGQ NO: 59) G (SEQ ID RKFQGRVTMTRDTSTSTVYM (SEQ ID NO: 65)APRLLIYGASTRATG (SEQ ID NO: 33) ELSSLRSEDTAVYYCARGGRT ID NO: NO:IPARFSGSGSGTEFT NO: 44) TWIGAFDIWGQGTMVTVSS 72) 69) LTISSLQSEDFAVYY(SEQ ID NO: 21) CQQYVVWPPLTFGG GTKVEIK (SEQ ID NO: 27) MAB19- VH1-YTFTSH VINPS ARLHV QVQLVQSGAEVKKPGASVKV VK3- RASQS GASTR QQYIVFEIVMTQSPATLSVSP IgG4 46 YMG MGATS SGSYY SCKASGYTFTSHYMGWVRQA 15 VSSNL ATPWT GERATLSCRASQSV (SEQ ID YAQKF PAYLD PGQGLEWMGV1NPSMGATSY A (SEQ(SEQ ID SSNLAWYQQKPGQ NO: 62) QG Y AQKFQGRVTMTRDTSTSTVY (SEQ ID NO: 66)APRHLIYGASTRAT (SEQ ID (SEQ ID MELSSLRSEDTAVYYCARLH ID NO: NO:GIPARFSGSGSGTEF NO: 45) NO: 35) VSGSYYPAYLDYWGQGTMV 72) 69)TLTISSLQSEDFAVY TVSS YCQQYIVFPWTFGG (SEQ ID NO: 22) GTKVEIK(SEQ ID NO: 28) MAB20- VH1- YTFTSH IINPSM ARLHV QVQLVQSGAEVKKPGASVKVVK3- RASQS GASTR QQYIVF EIVMTQSPATLSVSP IgG4 46 YMG GATSY SGSYYSCKASGYTFTSHYMGWVRQA 15 VSSNL AT PWT GERATLSCRASQSV (SEQ ID AQKFQ PAYLDPGQGLEWVGIINPSMGATSYA A (SEQ (SEQ ID SSNLAWYQQKPGQ NO: 62) G YQKFQGRVTMTRDTSTSTVYM (SEQ ID NO: 66) APRHLIYGASTRAT (SEQ ID (SEQ IDELSSLRSEDTAVYYCARLHVS ID NO: NO: GIPARFSGSGSGTEF NO: 46) NO: 35)GSYYPAYLDYWGQGTMVTV 72) 69) TLTISSLQSEDFAVY SS YCQQYIVFPWTFGG(SEQ ID NO: 23) GTKVEIK (SEQ ID NO: 28) MAB21- VH1- YTFTSH IINPSM ARLHVQVQLVQSGAEVKKPGASVKV VK3- RASQS GASTR QQYIVF EIVMTQSPATLSVSP IgG4 46 YMGGATSY SGSYY SCKASGYTFTSHYMGWVRQA 15 VSSNL AT PWT GERATLSCRASQSV (SEQ IDTQKFR PAYLD PGQGLEWMGIINPSMGATSYT A (SEQ (SEQ ID SSNLAWYQQKPGQ NO: 62) GY QKFRGRVTMTRDTSTSTVYM (SEQ ID NO: 66) APRHLIYGASTRAT (SEQ ID (SEQ IDELSSLRSEDTAVYYCARLHVS ID NO: NO: GIPARFSGSGSGTEF NO: 47) NO: 35)GSYYPAYLDYWGQGTMVTV 72) 69) TLTISSLQSEDFAVY SS YCQQYIVFPWTFGG(SEQ ID NO: 24) GTKVEIK (SEQ ID NO: 28) ¹Includes CDR-H1 as defined byboth the Chothia and Kabat numbering systems, inclusive of theboundaries of both numbering systems. ²According to the Kabat numberingsystem. ³According to the IMGT numbering system. ⁴According to the Kabatand Chothia numbering systems. ⁵According to the Kabat and Chothianumbering systems. ⁶According to the Kabat, Chothia, and IMGT numberingsystems.

Example 4: Antibody Characterization

ForteBio K_(D) Measurements:

Quantitative binding of antibodies to recombinant monomeric human, mouse(SEQ ID NO: 3), cynomolgus monkey TIGIT was measured using biolayerinterferometry (BLI) with a FORTEBIO®. Affinity measurements of selectedantibodies were performed generally as described in Estep et al., Mabs,2013, 5:270-278, incorporated by reference in its entirety. FORTEBIOaffinity measurements were performed by loading IgGs on-line onto AHQsensors. Sensors were equilibrated off-line in assay buffer for 30 minand then monitored on-line for 60 seconds for baseline establishment.Sensors with loaded IgGs were exposed to a single concentration ofantigen (100 nM) for 3 minutes. Afterwards they were transferred toassay buffer for 3 minutes for off-rate measurement. Kinetics wereanalyzed using the 1:1 binding model. A summary of K_(D) measurementsfor antibodies binding a single concentration of human, cynomolgusmonkey, and mouse (SEQ ID NO: 3) TIGIT is shown in Table 6 below.

MSD-SET K_(D) Measurements:

Solution equilibrium affinity measurements of selected antibodiesbinding to monomeric recombinant human and cynomolgus monkey TIGIT wereperformed generally as previously described. See Estep et al., supra,incorporated by reference in its entirety. Briefly, solution equilibriumtitrations (SET) were performed in PBS+0.1% IgG-Free BSA (PBSF) withantigen (TIGIT monomer) held constant at 10-100 pM and incubated with 3-to 5-fold serial dilutions of Fab or mAbs starting at 10 pM-10 nM.Antibodies (20 nM in PBS) were coated onto standard bind MSD-ECL platesovernight at 4° C. or at room temperature for 30 min. Plates were thenblocked by BSA for 30 min with shaking at 700 rpm, followed by threewashes with wash buffer (PBSF+0.05% Tween 20). SET samples were appliedand incubated on the plates for 150s with shaking at 700 rpm followed byone wash. Antigen captured on a plate was detected with 250 ng/mLsulfotag-labeled streptavidin in PBSF by incubation on the plate for 3min. The plates were washed three times with wash buffer and then readon the MSD Sector Imager 2400 instrument using 1× Read Buffer T withsurfactant. The percent free antigen was plotted as a function oftitrated antibody in Prism and fit to a quadratic equation to extractthe K_(D). To improve throughput, liquid handling robots were usedthroughout MSD-SET experiments, including SET sample preparation.

TABLE 6 K_(D) Measurements for Human, Cyno, and Mouse (SEQ ID NO: 3)TIGIT ForteBio ForteBio ForteBio MSD-SET MSD-SET K_(D) (M) Human K_(D)(M) Cyno K_(D) (M) Mouse K_(D) (M) Human K_(D) (M) Cyno Antibody TIGITHis TIGIT His TIGIT His TIGIT His TIGIT His MAB1 5.24E−10 2.64E−09 N.B.5.40E−11 3.20E−10 MAB2 4.57E−10 1.57E−09 N.B. 2.50E−11 2.30E−10 MAB33.32E−10 8.02E−10 N.B. 8.10E−12 3.50E−11 MAB4 2.46E−10 3.69E−10 N.B.5.00E−12 1.50E−11 MAB5 1.96E−10 8.98E−10 N.B. 4.90E−12 4.60E−11 MAB63.11E−09 1.75E−08 N.B. N.D N.D MAB7 2.54E−09 P.F. N.B. N.D N.D MAB83.13E−09 2.58E−08 N.B. N.D N.D MAB9 2.83E−09 9.35E−09 N.B. N.D N.D MAB101.71E−09 6.55E−09 P.F. 1.10E−10 N.D MAB11 2.47E−09 8.14E−09 N.B.1.50E−10 N.D MAB12 2.35E−09 6.57E−09 P.F. 5.60E−11 N.D MAB13 1.44E−09N.B. N.B. 4.00E−10 N.D MAB14 1.23E−09 N.B. N.B. 3.80E−10 N.D MAB155.26E−10 7.94E−08 N.B. 2.10E−10 N.D MAB16 3.78E−10 7.04E−08 N.B.7.00E−11 N.D MAB17 4.29E−10 1.10E−07 N.B. 4.10E−11 N.D MAB18 4.48E−107.20E−08 N.B. N.D N.D MAB19 P.F. N.B. N.B. N.D N.D MAB20 P.F. N.B. N.B.3.00E−11 N.D MAB21 P.F. N.B. N.B. 8.00E−11 N.D N.B.: Non-binder or weakbinder P.F.: Poor Fit (good binding response with unreportable K_(D)based on a 1:1 fitting model) N.D.: MSD affinity measurement was notperformed

Example 5: Evaluation of Blockade of TIGIT Ligands

Quantitative ligand blocking studies were conducted by using a cellsurface TIGIT binding assay. Binding of fluorescently labeled PVR-Fc orPVRL2-Fc to human TIGIT expressing Jurkat cells was measured by flowcytometry. A dilution series of each test antibody was incubated withthe TIGIT Jurkat cells in order to measure each antibody's ability toblock PVR-Fc or PVRL2-Fc binding and determine the IC₅₀ values shown inTable 7.

TABLE 7 Ligand blocking IC₅₀ Values for Antibody Panel PVR PVRL2Antibody IC₅₀ (nM) IC₅₀ (nM) MAB1-IgG4 2.2 1.4 MAB2-IgG4 2.3 1.3MAB3-IgG4 1.6 1.2 MAB4-IgG4 1.9 1.6 MAB5-IgG4 1.7 1.4 MAB6-IgG4 3.2 1.4MAB7-IgG4 2.6 2 MAB8-IgG4 2.9 1.2 MAB9-IgG4 1.9 1.1 MAB10-IgG4 3.3 1MAB11-IgG4 2 1.2 MAB12-IgG4 1.7 1.2 MAB13-IgG4 2.1 1.8 MAB14-IgG4 2.61.6 MAB15-IgG4 2.2 1.1 MAB16-IgG4 2.1 1.3 MAB17-IgG4 2.6 1.9 MAB18-IgG41.8 1.9 MAB19-IgG4 6.4 2 MAB20-IgG4 2.3 1.9 MAB21-IgG4 1 0.8

Example 6: Additional TIGIT Binding Assays to Measure Affinity andCross-Reactivity of Antibodies to TIGIT

The affinity of antibodies binding to human TIGIT was measured withmultiple concentrations of antigen in order to more accurately measurebinding kinetics. Additionally, quantitative binding of MAB10 to human,mouse (SEQ ID NO: 3), cynomolgus monkey TIGIT was measured usingbiolayer interferometry (BLI) and flow cytometry. FIG. 1A shows analignment of TIGIT from different species. The percent identities acrossthe whole TIGIT protein are summarized in Table 8 below.

TABLE 8 Percent identities between TIGIT proteins of different species.Cynomolgus Human Monkey Mouse Human 100 89.17 68.38 Cynomolgus Monkey89.17 100 66.67 Mouse 68.38 66.67 100Kinetic Measurements for Antibodies Binding to Human, Cynomolgus Monkey,and Mouse TIGIT

The binding affinities and kinetics for antibodies binding to humanTIGIT-His were measured using an Octet® QKe instrument (ForteBio) in amethod similar to that described above in Example 4 but with multipleconcentrations of antigen used. Additionally, binding of MAB10 tocynomolgus monkey and mouse (SEQ ID NO: 3) TIGIT-His was measured. Astrategy of capturing anti-TIGIT antibodies on sensors followingassociation/dissociation of monomeric TIGIT proteins was used to avoidavidity effects in the assay. The BLI analysis was performed at 29° C.using 1× kinetics buffer (ForteBio) as assay buffer. Anti-human IgG Fccapture (AHC) biosensors (ForteBio) were first presoaked in assay bufferfor over five minutes. Anti-TIGIT antibody (5 μg/mL) was captured on thesensor for 300 seconds. Sensors were then dipped in assay buffer for 120seconds to establish a baseline before measuring binding to each TIGITprotein. Sensors were then dipped into varying concentrations of humanTIGIT-His (12.4 to 0.8 nM or 6.2 to 0.8 nM, 2-fold dilutions in assaybuffer), cynomolgus monkey TIGIT-His (24.6 to 1.5 nM or 12.3 to 1.5 nM,2-fold dilutions in assay buffer, for MAB10 only), or mouse TIGIT-His(303 to 4.7 nM, 2-fold dilutions in assay buffer, for MAB10 only) for300 seconds or 600 seconds, depending on the experiment, to measureassociation. Dissociation of TIGIT was measured by dipping the sensorsinto assay buffer for 600, 1200, or 1800 seconds, depending on theexperiment (600 seconds was only used for mouse TIGIT-His). Agitation atall steps was 1000 rpm.

Kinetic parameters were generated with Octet® Data Analysis SoftwareVersion 8.2.0.7 using reference subtraction, dissociation basedinter-step correction, 1-to-1 binding model, and global fit (R_(max)unlinked by sensor). The association rate constant (k_(a)), dissociationrate constant (k_(d)) and equilibrium constant (K_(D)) values wereindividually averaged across experiments, and a summary of the data forantibodies binding to human TIGIT are shown in Table 9. A summary ofMAB10 binding to monomeric human, cynomolgus monkey, and mouse TIGIT(SEQ ID NO: 3) is shown in Table 10.

TABLE 9 TIGIT Antibody Multi-Concentration Kinetics for Binding HumanTIGIT Average of Average of Average of Antibody k_(a) (1/Ms) k_(d) (1/s)K_(D) (M) n MAB2 3.2E+05 2.3E−04 7.1E−10 2 MAB4 7.0E+05 6.3E−05 8.1E−113 MAB5 7.7E+05 1.4E−04 1.9E−10 2 MAB9 1.6E+06 8.5E−04 5.6E−10 2 MAB102.0E+06 3.8E−04 2.4E−10 6 MAB11 1.3E+06 3.5E−04 2.8E−10 2 MAB12 1.5E+062.4E−04 1.6E−10 2 MAB15 1.1E+06 6.6E−04 5.8E−10 2 MAB16 4.5E+05 3.5E−041.1E−09 3 MAB18 7.5E+05 5.9E−04 8.1E−10 3 MAB20 8.9E+05 3.8E−04 4.6E−102 MAB21 1.4E+06 5.0E−04 3.6E−10 2

TABLE 10 MAB10 Kinetic Parameters for Binding Human, Cynomolgus Monkey,and Mouse Average Average Average Species k_(a) (1/Ms) k_(d) (1/s) K_(D)(M) n Human 2.0E+06 3.8E−04 2.4E−10 6 Cynomolgus Monkey 7.9E+05 4.6E−036.2E−09 5 Mouse — — >7.0E−07* 3 *K_(D) could not be determined due tominimal binding (very low binding response), indicating that any bindingis poorer than the limit of the instrument's sensitivity.K_(D) Measurements for Binding to Cells Engineered to Express TIGIT

The K_(D) for MAB10 binding to cell surface TIGIT in engineered celllines was measured using flow cytometry. Jurkat cells (acute T cellleukemia, ATCC® TIB-152™) were engineered to stably express human orcynomolgus monkey TIGIT, and CHO-K1 cells were engineered to stablyexpress mouse TIGIT (SEQ ID NO: 3). The K_(D) values are shown in Table11. The K_(D) values for MAB10 binding to cell surface human andcynomolgus monkey TIGIT are very similar

TABLE 11 Measurement of K_(D) for MAB10 Binding to Cell Surface TIGIT onEngineered Cells Average Cell Line K_(D) (M) n Human TIGIT Jurkat5.1E−10 2 Cynomolgus Monkey TIGIT Jurkat 4.0E−10 1 Mouse TIGIT CHO-K19.8E−9  1K_(D) Measurements for Binding to Primary Cells

The K_(D) for MAB10 binding to cell surface TIGIT on primary cells wasmeasured using flow cytometry. For both human and cynomolgus monkeyPBMCs, CD8+ T cells had the greatest detectable TIGIT expression, andtherefore were used to calculate the binding of MAB10 to primary cellsin these species. For analysis purposes, CD8+ T cells were defined ascells with a lymphocyte size and granularity that expressed thefollowing combination of molecular markers: CD3+CD4−CD8+. Similarly,murine Tregs demonstrated the highest binding of MAB10, and weretherefore used for these calculations. Murine Tregs were defined asCD4+CD8−CD25+FoxP3+ cells of lymphocyte size and granularity. The K_(D)values are shown in Table 12. The K_(D) values for MAB10 binding to cellsurface human and cynomolgus monkey TIGIT on primary cells are verysimilar.

TABLE 12 Measurement of K_(D) for MAB10 Binding to Cell Surface TIGIT onPrimary Cells Average Cells K_(D) (M) n Human CD8 1.3E−9 2 CynomolgusMonkey CD8 2.8E−9 2 Mouse Tregs 2.5E−8 2Antibody Binding to Human PVRL4

In order to confirm the specificity of anti-TIGIT antibodies, binding tohuman PVRL4, the Ig family member most closely related to TIGIT (29%identity in extracellular region of homology), was measured by BLI. FIG.1B shows an alignment of the human TIGIT and PVRL4 extracellulardomains. The BLI analysis was performed at 30° C. using 1× kineticsbuffer as assay buffer. AHC sensors were first presoaked in assay bufferfor greater than 5 minutes. Antibody (5 μg/mL) was captured on thesensor for 300 seconds. Sensors were then dipped in assay buffer for 120seconds to establish a baseline before measuring binding to humanPVRL4-His protein. Sensors were then dipped into human PVRL4-His (200 nMin assay buffer) for 200 seconds to measure association. Dissociation ofPVRL4 was then measured by dipping sensors into assay buffer for 200seconds. Results were analyzed using Octet® Data Analysis SoftwareVersion 8.2.0.7. MAB1 through MAB21 did not bind PVRL4, thusdemonstrating that the MABs disclosed herein are highly specific forTIGIT.

Example 7: Production of IL-2 in Jurkat Cells Engineered to Respond toHuman TIGIT Signaling Following Treatment with Anti-TIGIT Antibodies

An assay for testing the ability of antibodies to inhibit the functionof TIGIT was developed using two engineered cell lines. This co-cultureassay was developed to mimic the interaction of a TIGIT expressing Tcell with a second cell expressing TIGIT ligand (PVR and PVRL2), thusreplicating TIGIT's ability to suppress T cell activation. Thisinteraction causes an inhibition of T cell function (e.g. cytokinerelease) in the TIGIT expressing cell. Jurkat cells (acute T cellleukemia) normally express IL-2 upon stimulation of the T cell receptor(using anti-CD3 and anti-CD28 agonist antibodies). The expression ofTIGIT in Jurkat cells would reduce IL-2 expression induced byanti-CD3/CD28 agonist antibodies if PVR and/or PVRL2 was present andbound to TIGIT, thus providing a suppressive signal to the Jurkat cell.Therefore, a Jurkat cell line was engineered to express human TIGIT.

A second cell line, HT-1080 (human fibrosarcoma cell line, ATCC®CCL121™), was engineered to express a membrane tethered anti-CD3 singlechain Fv (scFv) antibody that can provide an activating signal to theTIGIT Jurkat cells. The activating signal was also enhanced by includingsoluble anti-CD28 agonist antibody. HT-1080 cells naturally express highlevels of PVR and PVRL2, thus providing ligand for TIGIT in a TIGITJurkat/anti-CD3 HT-1080 co-culture assay. In this co-culture assay,TIGIT antagonist antibodies increase the production of IL-2 compared tonegative control antibodies. An overview of this assay system is shownin FIG. 3.

The co-culture assay was used to determine the EC₅₀ of anti-TIGITantibodies by treatment with a dose range of antibody. EC₅₀ was measuredfor anti-TIGIT antibodies MAB1-MAB21, as well as the hamster anti-mouseantibody SEC1 (see Example 8) and the commercial anti-human anti-TIGITantibody MBSA43 (available, e.g., from eBioscience, Cat. No. 16-9500).The supernatants were harvested as described above 24 hours posttreatment and analyzed by an IL-2 ELISA. A summary of the experimentallydetermined EC₅₀ values in human TIGIT Jurkat cells is in Table 13. Ascan be seen in Table 13, all of the MABs except for MAB13, MAB 14, MAB16, and SEC1, perform better in this assay than the commercial antibodyMBSA43.

TABLE 13 Average EC₅₀ Values in Human TIGIT Jurkat Co-Culture AssayAverage Antibody EC₅₀ (nM) MAB1 0.22 MAB2 0.31 MAB3 0.33 MAB4 0.34 MAB50.34 MAB6 data not available MAB7 0.25 MAB8 0.24 MAB9 0.06 MAB10 0.14MAB11 0.24 MAB12 0.16 MAB13 1.40 MAB14 0.71 MAB15 0.21 MAB16 1.11 MAB170.13 MAB18 0.25 MAB19 0.20 MAB20 0.68 MAB21 0.61 SEC1 (see below) 8.46MBSA43 0.45

Additionally, the Jurkat co-culture assay was repeated with MAB10 usinga subcloned isolate of the anti-CD3 scFv HT1080 cells. FIG. 4A shows theEC₅₀ curves from an exemplary experiment comparing MAB10 and an IgG4control. That experiment was conducted 3 times, and the average EC₅₀ was0.11 nM.

As described above for human TIGIT expressing Jurkat cells, a co-culturestimulation assay was set up with HT-1080 anti-CD3 scFv cells in thepresence of cynomolgus monkey TIGIT expressing Jurkat cells and solubleanti-human CD28. FIG. 4B shows the EC₅₀ curves from an exemplaryexperiment comparing MAB10 and IgG4 control. As shown in FIG. 4B, MAB10induces IL-2 production in cynomolgus monkey TIGIT expressing Jurkatcells, whereas the IgG4 isotype control does not. The average EC₅₀ forMAB10 in the cynomolgus monkey TIGIT Jurkat/anti-CD3 HT-1080 co-cultureassay was determined to be 2.87 nM.

Example 8: Characterization of the Anti-TIGIT Antibody “SEC1”

Additional studies were conducted to characterize the hamster anti-TIGITantibody 10A7 (disclosed, e.g., in U.S. Pat. Pub. No. 20090258013). Theantibody 10A7 was reformatted in two different ways for use in thisstudy. The first was to make a chimeric antibody with hamster variableregions and human IgG4 S228P (human S228P heavy chain, SEQ ID NO:73) andkappa constant regions (constant regions used for MAB10, SEQ ID NO:75).The second was to make a chimeric antibody with hamster variable regionsand mouse IgG2a N297A and kappa constant regions (heavy chain: SEQ IDNO:77; light chain: SEQ ID NO:79). The variable regions of theantibodies are provided in SEQ ID NOs: 74, 76, 78 and 80. Thereformatted 10A7 antibodies are referred to herein as “SEC1”.

Kinetic Measurements for SEC1 Binding to Recombinant Human, CynomolgusMonkey, and Mouse TIGIT

The binding affinities and kinetics of binding of SEC1 mouse IgG2a N297Ato human TIGIT-His, cynomolgus monkey TIGIT-His, and mouse (SEQ ID NO:3) TIGIT-His were measured using BLI with an Octet QKe instrument. Astrategy of capturing SEC1 on sensors followed byassociation/dissociation of monomeric TIGIT proteins was used to avoidavidity effects in the assay. The BLI analysis was performed at 29° C.using 1× Kinetics Buffer (ForteBio) as assay buffer. Anti-Mouse IgG FcCapture (AMC) biosensors (ForteBio) were first presoaked in assay bufferfor greater than 5 minutes. SEC1 mouse IgG2a N297A (5 μg/mL) wascaptured on the sensor for 300 seconds. Sensors were then dipped inassay buffer for 120 seconds to establish a baseline before measuringbinding to each TIGIT protein. Sensors were then dipped into varyingconcentrations of human TIGIT-His (33.8 to 1.25 nM, 3 fold dilutions inassay buffer), cynomolgus monkey TIGIT-His (302.8 to 0.42 nM, 3 folddilutions in assay buffer), or mouse TIGIT-His (33 to 1.22 nM, 3 folddilutions in assay buffer) for 300 seconds to measure association.Dissociation of TIGIT was then measured by dipping sensors into assaybuffer for 600 seconds. Agitation at all steps was 1000 rpm. Kineticparameters and sensorgrams were generated with Octet® Data AnalysisSoftware using reference subtraction, dissociation based inter-stepcorrection, 1 to 1 binding model, and global fit (Rmax unlinked bysensor). The K_(D) values are shown in Table 14.

TABLE 14 SEC1 IgG2a N297A Kinetic Parameters for Binding TIGIT Speciesk_(a) (1/Ms) k_(d) (1/s) K_(D) (M) Human 1.7E+06 7.9E−03 4.7E−09Cynomolgus Monkey No Binding Mouse 1.9E+06 6.0E−04 3.2E−10K_(D) Measurements for Binding to Primary Cells

The K_(D) for SEC1 (IgG4 S228P) binding to cell surface TIGIT on primarycells was measured using flow cytometry as described in Example 6. Forboth human and cynomolgus monkey PBMCs, CD8+ T cells had the greatestdetectable TIGIT expression, and therefore were used to calculate thebinding of SEC1 to primary cells in these species. For analysispurposes, CD8+ T cells were defined as cells with a lymphocyte size andgranularity that expressed the following combination of molecularmarkers: CD3+CD4−CD8+. Similarly, murine Tregs demonstrated the highestexpression of TIGIT, and were therefore used for these calculations.Murine Tregs were defined as CD4+CD8−CD25+FoxP3+ cells of lymphocytesize and granularity. The K_(D) values are shown in Table 15.

TABLE 15 Measurement of K_(D) for SEC1 Binding to Cell Surface TIGIT onPrimary Cells Average Cells K_(D) (M) n Human CD8 3.6E−9  1 CynomolgusMonkey CD8 No binding 1 Mouse Tregs 4.1E−10 2Engineered TIGIT Jurkat/Anti-CD3 HT-1080 Assay

SEC1 antagonizes TIGIT function in the engineered human TIGITJurkat/anti-CD3 HT-1080 co-culture assay described in Example 7. Theexperiment was conducted 3 times, and the average EC₅₀ was 8.5 nM. Incomparison, the average EC₅₀ for MAB10 in this assay is 0.14 nM.

Like MAB10, SEC1 has been described as a ligand blocking antibody, andboth antibodies inhibit the function of TIGIT in an engineered TIGITJurkat/anti-CD3 scFv HT-1080 co-culture assay.

Example 9: Increase in Cytokine Production in Sub-Optimally StimulatedHuman T Cells Following Treatment with MAB10

A study was developed in order to determine whether MAB10 is efficaciousin a cellular in vitro system using human primary T cells obtained fromhealthy donors. Two different forms of the assay were used: stimulationof T cells within a mix of PBMCs and stimulation of CD4+ T cells afterisolation from PBMCs. TIGIT is expressed in exhausted intra-tumoral CD8+T cells, NK and regulatory T cells. This study was designed to identifyand obtain more readily available TIGIT expressing human primary T cellsto use as a surrogate system for the intra-tumoral target cells.

In CD4+ T cells, TIGIT expression is primarily restricted to memorycells (CD45RO+). Suboptimal stimulation of CD4+ T cells permits assayingthe efficacy of MAB10 by measuring the increased production of IFN-γfollowing inhibition of TIGIT-ligand interactions.

Human primary T cells were obtained from healthy donors. Totalperipheral blood mononuclear cells (PBMCs) were isolated fromleukapheresis preparations and CD4+ T cells were in turn isolated fromPBMCs.

PBMCs were purified using Ficoll® density gradients. PBMCs were thenused to purify CD4+ cells using negative selection (CD4 T cell isolationkit, Miltenyi) following manufacturer's protocol.

FACS Analysis of Key Markers on Human CD4+ T Cells.

CD4+ T cells were sub-optimally stimulated with plate-bound anti-CD3antibody (1 μg/mL) and soluble anti-CD28 antibody (2 μg/mL) for 60hours. For staining and FACS analysis, cells from unstimulated andstimulated samples were used. The following antibodies were used forstaining: anti-TIGIT-PE-Cy7, anti-PVR-PE, anti-CD4-APC-eFluor780 andanti-CD45RA-APC, anti-CD45RO PerCP-eFluor710, and CD226-FITC. Cells wereanalyzed by flow cytometry using a BD LSRFortessa™ instrument.

Sub-optimal stimulation of PBMCs was achieved by addition of lowconcentrations of anti-CD3 antibody (0.2 μg/mL). Sub-optimal stimulationof CD4+ T cells was achieved by culturing the cells on 96-wellflat-bottom plates that had been previously coated with 1 μg/mL ofanti-CD3 antibody and 2 μg/mL soluble anti-CD28 antibody. After 60 hoursof culture, supernatants were collected and frozen for cytokinequantification using ELISA, AlphaLISA® or multiplex/Luminex® technology.The effect of MAB10 addition was compared to the addition of anon-specific control IgG4 antibody.

Purified CD4+ T cells were left unstimulated or were stimulated for 60hours using plate-bound anti-CD3 antibody (1 μg/mL) and solubleanti-CD28 antibody (2 μg/mL). FACS analysis was performed on bothunstimulated (FIG. 5A) and stimulated cells (FIG. 5B). Populationpercentages and mean fluorescence intensity (MFI) were calculated.Expression analysis of cell markers TIGIT, PVR, and CD226 prior andpost-activation showed that the percentage of both PVR and CD226positive cells increases upon activation. For TIGIT, the percentage ofTIGIT positive cells only increased moderately with these activationconditions, but the MFI values indicated a clear upregulation of TIGITexpression in the positive cell population. FACS analysis also confirmedthat TIGIT expression was restricted primarily to memory cells(CD45RO+). CD4+ T cells from a representative donor were stained forCD45RA (naïve T cell marker) and CD45RO (activated or memory T cellmarker) markers to differentiate naïve and memory T cells. Expressionlevels of TIGIT were analyzed within each of these populations (see FIG.5C).

Purified PBMCs obtained from healthy donors were stimulated for 60 hoursusing soluble anti-CD3 antibody (0.2 μg/mL) in the presence of differentconcentrations of a MAB or a control IgG4 antibody. Cell culturesupernatants were collected and used to measure production ofpro-inflammatory cytokines. The analysis of samples from two humandonors, illustrated in FIGS. 6A-6K, show that treatment with each of theMABs induces the upregulation IFN-γ. MAB10 was then used to induce theproduction of several pro-inflammatory cytokines in PBMCs from Donor 1,including tumor necrosis factor alpha (TNF, FIG. 6L), lymphotoxin alpha(LT-α, FIG. 6M), and interferon gamma (IFN-γ, FIG. 6N). A graphicalanalysis of the EC₅₀ for IFN-γ is shown in FIG. 6O. PBMCs from Donor 2were similarly treated with MAB10 and cytokines induced as shown in FIG.7A (IFN-γ), FIG. 7B (TNF), FIG. 7C (IL-6), FIG. 7D (GM-CSF), and FIG. 7E(LT-α). EC₅₀ value for MAB10 in this assay in the two donors tested wasaveraged as ˜16 nM, by determining the concentration of MAB10 requiredto induce 50% of the increase in IFN-γ, TNF and LT-αsignal. A summary ofthe TNF data for the two donors (FIG. 6) is shown in Table 16.

TABLE 16 Data Summary for Two Donors (TNF Analyzed) Donor EC₁₀ (nM) EC₅₀(nM) EC₉₀ (nM) 1 5.02 12.60 31.59 2 18.86 20.60 22.49 Average 11.9416.60 27.04

Purified CD4+ T cells obtained from 3 different healthy donors werestimulated for 60 hours using plate-bound anti-CD3 antibody (1 μg/mL)and soluble anti-CD28 antibody (2 μg/mL) in the presence of differentconcentrations of a control IgG4 antibody or MAB10. Cell culturesupernatants were collected and used to measure levels of IFN-γproduction.

In these sub-optimally stimulated CD4+ T cells, MAB10 addition resultsin an upregulation of IFN-γ in a dose dependent manner in all threedonors, demonstrating the anti-TIGIT antagonistic function of MAB10 (seeFIG. 8A (Donor 1), FIG. 8B (Donor 2), and FIG. 8C (Donor 3)). IFN-γproduction in cells treated with either MAB10 (black bars) or the IgG4isotype control (light gray bars) is shown in the left panel of eachFigure. EC₅₀ average value for MAB10 in this assay was calculated as1.02 nM by determining the concentration of MAB10 required to induce 50%of the increase in IFN-γ signal (plotted in the right panel of each ofFIGS. 8A-8C). The data are summarized in Table 17.

TABLE 17 Data Summary for Three Donors Donor EC₁₀ (nM) EC₅₀ (nM) EC₉₀(nM) 1 0.37 1.00 2.72 2 0.85 0.94 1.04 3 1.05 1.12 1.19 Average 0.751.02 1.65

As described above, addition of MAB10 to sub-optimally stimulated humanT cells antagonizes TIGIT function and induces the upregulation ofpro-inflammatory cytokines (e.g., IFN-γ and TNF) when compared to anon-specific control IgG4 antibody. This effect is dose-dependent withan estimated EC₅₀ of 1 nM for the isolated CD4+ T cell assay. These datademonstrate in vitro efficacy of MAB10 in normal primary human T cells.

Example 10: Characterization of MAB10 in PD-1/TIGIT Combination Bioassay

PD-1 is an immune inhibitory receptor expressed on activated T cells andB cells and plays a critical role in regulating immune responses totumor antigens and autoantigens. Engagement of PD-1 by either of itsligands, PD-L1 or PD-L2, on an adjacent cell inhibits T-cell receptor(TCR) signaling and TCR-mediated proliferation, transcriptionalactivation and cytokine production. Therapeutic antibodies and Fc fusionproteins designed to block the PD-1/PD-L1 interaction show promisingresults in clinical trials for the treatment of a variety of cancers.

The PD-1/TIGIT Combination Bioassay (Promega) is a biologically relevantmechanism of action-based assay that can be used to measure the potencyand stability of antibodies and other biologics designed to block thePD-1/PD-L1 and TIGIT/CD155 interactions in combination. The assayconsists of two genetically engineered cell lines: PD-1/TIGIT EffectorCells, which are Jurkat T-cells stably expressing human PD-1, TIGIT, anda luciferase reporter, and PD-L1/CD155 APC/CHO-K1 Cells, which areCHO-K1 cells stably expressing human PD-L1, human CD155, and a cellsurface protein (in this case, TIGIT) designed to activate cognate TCRsin an antigen-independent manner.

When the two cell types are co-cultured, the PD-1/PD-L1 and TIGIT/CD155interactions inhibit TCR signaling and luciferase activity. Addition ofan antibody, e.g., an ABP disclosed herein or known in the art, thatbinds TIGIT and blocks ligand binding (e.g., CD155), in combination witha second antibody that blocks the interaction of PD-1 with its ligand(e.g., PD-L1), releases the inhibitory signal and results in TCRsignaling and NFAT-mediated luciferase activity.

FIG. 9A shows the results of the assay in which a 1:1 ratio of MAB10 andpembrolizumab (anti-PD-1 antibody) was used. Concentrations for eachantibody were 25, 10, 4, 1.6, 0.64, 0.256, 0.1024, 0.04096, and 0.016384μg/ml. An untargeted IgG4 was used as a control. As shown in the Figure,only the combination of MAB10 and pembrolizumab (EC₅₀ of 5.06 nM)blocked binding sufficiently to induce luciferase activity in the Jurkatcells. Neither the IgG4 control alone or the IgG4+MAB10 combinationinduced luciferase activity.

The assay was then repeated with a 1 μg/ml fixed dose of pembrolizumab(and a 1 μg/ml fixed dose of the IgG4 control) and a varying dose ofMAB10 (50, 20, 8, 3.2, 1.28, 0.512, 0.2048, 0.08192, and 0.032768μg/ml). As shown in FIG. 9B, while the fixed dose of pembrolizumabresulted in a low level of activation of luciferase induction, thecombination of pembrolizumab and MAB10 was much more effective atinduction of luciferase, with an EC₅₀ of 0.78 nM. As in FIG. 9A, neitherthe IgG4 control alone or the IgG4+MAB10 combination induced luciferaseactivity.

Example 11: Combination Therapy of CMV+ T-Cells with MAB10 andPembrolizumab

A lymphoproliferation assay was used to test for T-cell responses incytomegalovirus positive (CMV+) T-cells. PBMCs from individual donorsthat have been screened for CMV antigen reactivity were purchased fromAstarte Biologics (Bothell, Wash.). Cell lysates from CMV-infected cellswere also purchased from Astarte Biologics. The PBMCs were plated andthe antigen-specific stimulation is performed by the addition of celllysate, which stimulates the CMV+ T-cells in the sample. MAB10, an IgG4control, and/or the anti-PD-1 antibody pembrolizumab were added. Cellswere cultured for five days, and the supernatants were collected andanalyzed for the production of the effector cytokine TNF. Further datawere collected performing intracellular cytokine staining for othereffector molecules including IL-2, IFN-γ, perforin and granzyme-B.

Cells from a single donor (Donor 1) were stimulated and cultured asdescribed above. As shown in FIG. 10, by gating on CD4+ cells,incubation with MAB10 (black bars) increases the production of theeffector cytokines in a dose-dependent matter, as measured byintracellular staining, including TNF (FIG. 10A), IL-2 (FIG. 10B), andIFN-γ (FIG. 10C) to a greater extent than cells incubated with the IgG4control (white bars). Incubation with MAB10 also increases theproportion of antigen-specific activated CD4+ T-cells, as shown in FIG.10D, in which cells that were treated with 20 μg/ml of the IgG4 controlor MAB10 were analyzed by FACS by expression of CD3 (a marker of matureT-cells) and expression of TNF and IL-2.

Similar results were obtained by gating on CD8+ cells. As shown in FIG.11, on gated CD8+ cells, incubation with MAB10 (black bars) increasesthe production of the effector cytokines in a dose-dependent matter,including TNF (FIG. 11A), perforin (FIG. 11B), and granzyme B (FIG. 11C)compared to cells incubated with the IgG4 control (white bars). Perforinand granzyme B are markers of activated cytotoxic T-lymphocytes.Incubation with MAB10 also increases the proportion of antigen-specificactivated CD8+ T-cells, as shown in FIG. 11D. Cells that were treatedwith 20 μg/ml of the IgG4 control or MAB10 were analyzed by FACS byexpression of CD3 and expression of perforin and granzyme B.

Cells from the same donor were used in a similar set of experiments toshow that blockade by MAB10 amplifies CMV-specific CD8+ T-cellresponses. Cells were incubated with a range of concentrations of MAB10(black bars) or the IgG4 control (white bars) and the percentage ofdouble positive population perforin+granzyme B+(FIG. 12A) or IFN-γ+TNF+(FIG. 12C) was analyzed. FIGS. 12B (perforin+granzyme B+ analysis)and 12D (IFN-γ+ TNF+ analysis) show the proportion of double positivecells comparing cells treated with 20 μg/ml of the control antibody(left panels) or 20 μg/ml of MAB10 (right panels). The cells treatedwith MAB10 showed a much greater production of effector cytokinescompared with control-treated cells.

The combinatorial effect of MAB10 and the PD-1 antibody pembrolizumabwas tested using the same donor as described above. Cells werestimulated with CMV lysates as described above and treated with 2 μg/mlpembrolizumab or control IgG4, and 10, 20, or 40 μg/ml control antibodyor MAB10, and production of TNF in the supernatant was measured. Asshown in FIG. 13, four groups of cells were tested, treated with IgG4control (white bars, left most group), a constant amount of IgG4 controland a titration of MAB10 (dark gray bars, second group from left), aconstant amount of pembrolizumab and a titration of IgG4 control (lightgray bars, second group from right), or a constant amount ofpembrolizumab and a titration of MAB10 (black bars, right hand group).The combination of pembrolizumab and MAB10 increased the production ofTNF above the effect observed with the single agents.

The combination was again tested in three different donors, using theassay described above. Cells were stimulated with CMV lysate and treatedwith 20 μg/ml of MAB10 or 20 μg/ml of control IgG4 antibody and atitration of pembrolizumab, and production of TNF was measured. As shownin FIG. 14A (Donor 1), FIG. 14B (Donor 2), and FIG. 14C (Donor 3), theaddition of MAB10 (black bars), alone or in combination with increasingconcentrations of pembrolizumab, results in greater production of TNFcompared to the control antibody+pembrolizumab group (white bars).Additionally, MAB10 (black bars) in combination with pembrolizumab alsoresulted in increased activation compared to MAB10 alone. Statisticaldifferences were calculated between MAB10 alone and MAB10+pembrolizumabgroups using Student T test analysis (*=p<0.05, **=p<0.01, ***=p<0.005,****=p<0.001)

Taken together, the data presented in the Example demonstrate a clear,dose-dependent effect of MAB10 as a single agent in antigen-specificrecall assays. In addition, the data show increased efficacy whencombining MAB10 and pembrolizumab in multiple donors, indicating thevalue of the ABPs disclosed herein and PD-1 inhibitors or PD-L1inhibitors as combination therapies.

INCORPORATION BY REFERENCE

The entire disclosures of all patent and non-patent publications citedherein are each incorporated by reference in their entireties for allpurposes.

Other Embodiments

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in this application, in applications claiming priority fromthis application, or in related applications. Such claims, whetherdirected to a different invention or to the same invention, and whetherbroader, narrower, equal, or different in scope in comparison to theoriginal claims, also are regarded as included within the subject matterof the inventions of the present disclosure.

APPENDIX A SEQUENCE REFERENCE TABLE SEQ ID NO Molecule Region Sequence 1hTIGIT MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGED CAELHDYFNVLSYRSLGNCSFFTETG 2 cTIGITMRWCLFLIWAQGLRQAPLASGMMTGTIETTGNISAK KGGSVILQCHLSSTMAQVTQVNWEQHDHSLLAIRNAELGWHIYPAFKDRVAPGPGLGLTLQSLTMNDTGEYFCTYHTYPDGTYRGRIFLEVLESSVAEHSARFQIPLLGAMAMMLVVICIAVIVVVVLARKKKSLRIHSVESGLQRKSTGQEEQIPSAPSPPGSCVQAEAAPAGLCGEQQ GDDCAELHDYFNVLSYRSLGSCSFFTETG 3mTIGIT MHGWLLLVWVQGLIQAAFLATGATAGTIDTKRNISAEEGGSVILQCHFSSDTAEVTQVDWKQQDQLLAIYS VDLGWHVASVFSDRVVPGPSLGLTFQSLTMNDTGEYFCTYHTYPGGIYKGRIFLKVQESSVAQFQTAPLGGTMAAVLGLICLMVTGVTVLARKKSIRMHSIESGLGRTEAEPQEWNLRSLSSPGSPVQTQTAPAGPCGEQAEDD YADPQEYFNVLSYRSLESFIAVSKTG 4 MAB1-VH QLQLQESGPGLVKPSETLSLTCTVSGGSITSSSYYWG IgG4WIRQPPGKGLEWIGSIYYSGATFYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGSAWA FDPWGQGTLVTVSS 5 MAB2- VHQLQLQESGPGLVKPSETLSLTCTVSGGSISSSKYYWG IgG4WIRQPPGKGLEWIGSIYYSGSTFYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGSAWAF DPWGQGTLVTVSS 6 MAB3- VHQLQLQESGPGLVKPSETLSLTCTVSGGSISSTSHYWG IgG4WIRQPPGKGLEWIGSIYYSGSTFYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGSAWAF DPWGQGTLVTVSS 7 MAB4- VHQLQLQESGPGLVKPSETLSLTCTVSGGSISSTSHYWG IgG4WIRQPPGKGLEWIGSIYYSGSTFYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGGAWAF DPWGQGTLVTVSS 8 MAB5- VHQLQLQESGPGLVKPSETLSLTCTVSGGSISSTSHYWG IgG4WIRQPPGKGLEWIGSIYYSGSTFYNPSLKGRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGSAWA FDPWGQGTLVTVSS 9 MAB6- VHQLQLQESGPGLVKPSETLSLTCTVSGGSIESGSYYWG IgG4WIRQPPGKGLEWIGSIYYSGGTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLTLNKRSF DIVVGQGTMVTVSS 10 MAB7- VHQVQLQESGPGLVKPSQTLSLTCTVSGGSIESGVYYW IgG4GWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLTLNKRS FDIWGQGTMVTVSS 11 MAB8- VHQLQLQESGPGLVKPSETLSLTCTVSGGSIASGSYYWG IgG4WIRQPPGKGLEWIGSIYYSGQTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLTLNKRSF DIWGQGTMVTVSS 12 MAB9- VHQVQLQESGPGLVKPSQTLSLTCTVSGGSIESGLYYW IgG4GWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLTLNKRS FDIWGQGTMVTVSS 13 MAB10- VHQVQLQESGPGLVKPSQTLSLTCTVSGGSIESGLYYW IgG4GWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLALNKR SFDIWGQGTMVTVSS 14 MAB11- VHQVQLQESGPGLVKPSQTLSLTCTVSGGSIESGLYYW IgG4GWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLALNKR SFDIWGQGTMVTVSS 15 MAB12- VHQVQLQESGPGLVKPSQTLSLTCTASGGSIESGLYYW IgG4GWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLALNKR SFDIWGQGTMVTVSS 16 MAB13- VHQVQLVQSGAEVKKPGASVKVSCKASGYTFGNYYM IgG4HWVRQAPGQGLEWMGIINPSLGLTSYAQKFQGRVT MTRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGAFDIWGQGTMVTVSS 17 MAB14- VH QVQLVQSGAEVKKPGASVKVSCKASGYTFPAYYMH IgG4WVRQAPGQGLEWMGIINPSLGLTSYAQKFQGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGAFDIWGQGTMVTVSS 18 MAB15- VH QVQLVQSGAEVKKPGASVKVSCKASGYTFREYYMH IgG4WVRQAPGQGLEWMGIINPSIGLTSYARKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGA FDIWGQGTMVTVSS 19 MAB16- VHQVQLVQSGAEVKKPGASVKVSCKASGYTFREYYMH IgG4WVRQAPGQGLEWMGIINPSIGLTSYARKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGA LDIWGQGTMVTVSS 20 MAB17- VHQVQLVQSGAEVKKPGASVKVSCKASGYTFPAYYIH IgG4WVRQAPGQGLEWMGIINPSLGLTSYARKFQGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGALDIWGQGTMVTVSS 21 MAB18- VH QVQLVQSGAEVKKPGASVKVSCKASGYTFPAYYMH IgG4WVRQAPGQGLEWMGIINPSLGLTSYARKFQGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGAFDIWGQGTMVTVSS 22 MAB19- VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHYMG IgG4WVRQAPGQGLEWMGVINPSMGATSYAQKFQGRVT MTRDTSTSTVYMELSSLRSEDTAVYYCARLHVSGSYYPAYLDYWGQGTMVTVSS 23 MAB20- VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHYMG IgG4WVRQAPGQGLEWVGIINPSMGATSYAQKFQGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCARLHVSGSYYPAYLDYWGQGTMVTVSS 24 MAB21- VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHYMG IgG4WVRQAPGQGLEWMGIINPSMGATSYTQKFRGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCARLHVSGSYYPAYLDYWGQGTMVTVSS 25 MAB1- VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ IgG4QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQQHFNLPTFGGGTKVEIK25 MAB2- VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ IgG4QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQQHFNLPTFGGGTKVEIK25 MAB3- VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ IgG4QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQQHFNLPTFGGGTKVEIK25 MAB4- VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ IgG4QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQQHFNLPTFGGGTKVEIK25 MAB5- VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ IgG4QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQQHFNLPTFGGGTKVEIK26 MAB6- VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY IgG4QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTKVEIK 26 MAB7- VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY IgG4QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTKVEIK 26 MAB8- VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY IgG4QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTKVEIK 26 MAB9- VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY IgG4QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTKVEIK 26 MAB10- VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY IgG4QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTKVEIK 26 MAB11- VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY IgG4QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTKVEIK 26 MAB12- VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY IgG4QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTKVEIK 27 MAB13- VLEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY IgG4QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYVVWPPLTFGGGTKVEIK 27 MAB14- VLEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY IgG4QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYVVWPPLTFGGGTKVEIK 27 MAB15- VLEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY IgG4QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYVVWPPLTFGGGTKVEIK 27 MAB16- VLEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY IgG4QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYVVWPPLTFGGGTKVEIK 27 MAB17- VLEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY IgG4QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYVVWPPLTFGGGTKVEIK 27 MAB18- VLEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY IgG4QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYVVWPPLTFGGGTKVEIK 28 MAB19- VLEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY IgG4QQKPGQAPRHLIYGASTRATGIPARFSGSGSGTEFTL TISSLQSEDFAVYYCQQYIVFPWTFGGGTKVEIK28 MAB20- VL EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY IgG4QQKPGQAPRHLIYGASTRATGIPARFSGSGSGTEFTL TISSLQSEDFAVYYCQQYIVFPWTFGGGTKVEIK28 MAB21- VL EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY IgG4QQKPGQAPRHLIYGASTRATGIPARFSGSGSGTEFTL TISSLQSEDFAVYYCQQYIVFPWTFGGGTKVEIK29 MAB1- H3-IMGT ARDANYYGSAWAFDP IgG4 29 MAB2- H3-IMGT ARDANYYGSAWAFDPIgG4 29 MAB3- H3-IMGT ARDANYYGSAWAFDP IgG4 30 MAB4- H3-IMGTARDANYYGGAWAFDP IgG4 29 MAB5- H3-IMGT ARDANYYGSAWAFDP IgG4 31 MAB6-H3-IMGT ARDGVLTLNKRSFDI IgG4 31 MAB7- H3-IMGT ARDGVLTLNKRSFDI IgG4 31MAB8- H3-IMGT ARDGVLTLNKRSFDI IgG4 31 MAB9- H3-IMGT ARDGVLTLNKRSFDI IgG432 MAB10- H3-IMGT ARDGVLALNKRSFDI IgG4 32 MAB11- H3-IMGT ARDGVLALNKRSFDIIgG4 32 MAB12- H3-IMGT ARDGVLALNKRSFDI IgG4 33 MAB13- H3-IMGTARGGRTTWIGAFDI IgG4 33 MAB14- H3-IMGT ARGGRTTWIGAFDI IgG4 33 MAB15-H3-IMGT ARGGRTTWIGAFDI IgG4 34 MAB16- H3-IMGT ARGGRTTWIGALDI IgG4 34MAB17- H3-IMGT ARGGRTTWIGALDI IgG4 33 MAB18- H3-IMGT ARGGRTTWIGAFDI IgG435 MAB19- H3-IMGT ARLHVSGSYYPAYLDY IgG4 35 MAB20- H3-IMGTARLHVSGSYYPAYLDY IgG4 35 MAB21- H3-IMGT ARLHVSGSYYPAYLDY IgG4 36 MAB1-H2-Kabat SIYYSGATFYNPSLKS IgG4 37 MAB2- H2-Kabat SIYYSGSTFYNPSLKS IgG437 MAB3- H2-Kabat SIYYSGSTFYNPSLKS IgG4 37 MAB4- H2-KabatSIYYSGSTFYNPSLKS IgG4 38 MAB5- H2-Kabat SIYYSGSTFYNPSLKG IgG4 39 MAB6-H2-Kabat SIYYSGGTYYNPSLKS IgG4 40 MAB7- H2-Kabat SIYYSGSTYYNPSLKS IgG441 MAB8- H2-Kabat SIYYSGQTYYNPSLKS IgG4 40 MAB9- H2-KabatSIYYSGSTYYNPSLKS IgG4 40 MAB10- H2-Kabat SIYYSGSTYYNPSLKS IgG4 40 MAB11-H2-Kabat SIYYSGSTYYNPSLKS IgG4 40 MAB12- H2-Kabat SIYYSGSTYYNPSLKS IgG442 MAB13- H2-Kabat IINPSLGLTSYAQKFQG IgG4 42 MAB14- H2-KabatIINPSLGLTSYAQKFQG IgG4 43 MAB15- H2-Kabat IINPSIGLTSYARKFQG IgG4 43MAB16- H2-Kabat IINPSIGLTSYARKFQG IgG4 44 MAB17- H2-KabatIINPSLGLTSYARKFQG IgG4 44 MAB18- H2-Kabat IINPSLGLTSYARKFQG IgG4 45MAB19- H2-Kabat VINPSMGATSYAQKFQG IgG4 46 MAB20- H2-KabatIINPSMGATSYAQKFQG IgG4 47 MAB21- H2-Kabat IINPSMGATSYTQKFRG IgG4 48MAB1- H1 -Chothia + GSITSSSYYWG IgG4 Kabat 49 MAB2- H1-Chothia +GSISSSKYYWG IgG4 Kabat 50 MAB3- H1-Chothia + GSISSTSHYWG IgG4 Kabat 50MAB4- H1-Chothia + GSISSTSHYWG IgG4 Kabat 50 MAB5- H1-Chothia +GSISSTSHYWG IgG4 Kabat 51 MAB6- H1-Chothia + GSIESGSYYWG IgG4 Kabat 52MAB7- H1-Chothia + GSIESGVYYWG IgG4 Kabat 53 MAB8- H1-Chothia +GSIASGSYYWG IgG4 Kabat 54 MAB9- H1-Chothia + GSIESGLYYWG IgG4 Kabat 54MAB10- H1-Chothia + GSIESGLYYWG IgG4 Kabat 54 MAB11- H1-Chothia +GSIESGLYYWG IgG4 Kabat 54 MAB12- H1-Chothia + GSIESGLYYWG IgG4 Kabat 55IgG4 Constant, S228P ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVhinge stabilizing TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K 56 IgG4 Constant S228P,ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV N297A, CTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS terminal LysSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP deletedAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 57 IgG1 ConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV (G1m(3)TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS allotype)SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 58 MAB13- H1-Chothia +YTFGNYYMH IgG4 Kabat 59 MAB14- H1-Chothia + YTFPAYYMH IgG4 Kabat 60MAB15- H1-Chothia + YTFREYYMH IgG4 Kabat 60 MAB16- H1-Chothia +YTFREYYMH IgG4 Kabat 61 MAB17- H1-Chothia + YTFPAYYIH IgG4 Kabat 59MAB18- H1-Chothia + YTFPAYYMH IgG4 Kabat 62 MAB19- H1-Chothia +YTFTSHYMG IgG4 Kabat 62 MAB20- H1-Chothia + YTFTSHYMG IgG4 Kabat 62MAB21- H1-Chothia + YTFTSHYMG IgG4 Kabat 63 MAB1- L3- QQHFNLPT IgG4Chothia/Kabat/ IMGT 63 MAB2- L3- QQHFNLPT IgG4 Chothia/Kabat/ IMGT 63MAB3- L3- QQHFNLPT IgG4 Chothia/Kabat/ IMGT 63 MAB4- L3- QQHFNLPT IgG4Chothia/Kabat/ IMGT 63 MAB5- L3- QQHFNLPT IgG4 Chothia/Kabat/ IMGT 64MAB6- L3- QQHTVRPPLT IgG4 Chothia/Kabat/ IMGT 64 MAB7- L3- QQHTVRPPLTIgG4 Chothia/Kabat/ IMGT 64 MAB8- L3- QQHTVRPPLT IgG4 Chothia/Kabat/IMGT 64 MAB9- L3- QQHTVRPPLT IgG4 Chothia/Kabat/ IMGT 64 MAB10- L3-QQHTVRPPLT IgG4 Chothia/Kabat/ IMGT 64 MAB11- L3- QQHTVRPPLT IgG4Chothia/Kabat/ IMGT 64 MAB12- L3- QQHTVRPPLT IgG4 Chothia/Kabat/ IMGT 65MAB13- L3- QQYVVWPPLT IgG4 Chothia/Kabat/ IMGT 65 MAB14- L3- QQYVVWPPLTIgG4 Chothia/Kabat/ IMGT 65 MAB15- L3- QQYVVWPPLT IgG4 Chothia/Kabat/IMGT 65 MAB16- L3- QQYVVWPPLT IgG4 Chothia/Kabat/ IMGT 65 MAB17- L3-QQYVVWPPLT IgG4 Chothia/Kabat/ IMGT 65 MAB18- L3- QQYVVWPPLT IgG4Chothia/Kabat/ IMGT 66 MAB19- L3- QQYIVFPWT IgG4 Chothia/Kabat/ IMGT 66MAB20- L3- QQYIVFPWT IgG4 Chothia/Kabat/ IMGT 66 MAB21- L3- QQYIVFPWTIgG4 Chothia/Kabat/ IMGT 67 MAB1- L2- DASNRAT IgG4 Chothia/Kabat 67MAB2- L2- DASNRAT IgG4 Chothia/Kabat 67 MAB3- L2- DASNRAT IgG4Chothia/Kabat 67 MAB4- L2- DASNRAT IgG4 Chothia/Kabat 67 MAB5- L2-DASNRAT IgG4 Chothia/Kabat 68 MAB6- L2- GASSRAT IgG4 Chothia/Kabat 68MAB7- L2- GASSRAT IgG4 Chothia/Kabat 68 MAB8- L2- GASSRAT IgG4Chothia/Kabat 68 MAB9- L2- GASSRAT IgG4 Chothia/Kabat 68 MAB10- L2-GASSRAT IgG4 Chothia/Kabat 68 MAB11- L2- GASSRAT IgG4 Chothia/Kabat 68MAB12- L2- GASSRAT IgG4 Chothia/Kabat 69 MAB13- L2- GASTRAT IgG4Chothia/Kabat 69 MAB14- L2- GASTRAT IgG4 Chothia/Kabat 69 MAB15- L2-GASTRAT IgG4 Chothia/Kabat 69 MAB16- L2- GASTRAT IgG4 Chothia/Kabat 69MAB17- L2- GASTRAT IgG4 Chothia/Kabat 69 MAB18- L2- GASTRAT IgG4Chothia/Kabat 69 MAB19- L2- GASTRAT IgG4 Chothia/Kabat 69 MAB20- L2-GASTRAT IgG4 Chothia/Kabat 69 MAB21- L2- GASTRAT IgG4 Chothia/Kabat 70MAB1- L1- RASQSVSSYLA IgG4 Chothia/Kabat 70 MAB2- L1- RASQSVSSYLA IgG4Chothia/Kabat 70 MAB3- L1- RASQSVSSYLA IgG4 Chothia/Kabat 70 MAB4- L1-RASQSVSSYLA IgG4 Chothia/Kabat 70 MAB5- L1- RASQSVSSYLA IgG4Chothia/Kabat 71 MAB6- L1- RASQSVSSSYLA IgG4 Chothia/Kabat 71 MAB7- L1-RASQSVSSSYLA IgG4 Chothia/Kabat 71 MAB8- L1- RASQSVSSSYLA IgG4Chothia/Kabat 71 MAB9- L1- RASQSVSSSYLA IgG4 Chothia/Kabat 71 MAB10- L1-RASQSVSSSYLA IgG4 Chothia/Kabat 71 MAB11- L1- RASQSVSSSYLA IgG4Chothia/Kabat 71 MAB12- L1- RASQSVSSSYLA IgG4 Chothia/Kabat 72 MAB13-L1- RASQSVSSNLA IgG4 Chothia/Kabat 72 MAB14- L1- RASQSVSSNLA IgG4Chothia/Kabat 72 MAB15- L1- RASQSVSSNLA IgG4 Chothia/Kabat 72 MAB16- L1-RASQSVSSNLA IgG4 Chothia/Kabat 72 MAB17- L1- RASQSVSSNLA IgG4Chothia/Kabat 72 MAB18- L1- RASQSVSSNLA IgG4 Chothia/Kabat 72 MAB19- L1-RASQSVSSNLA IgG4 Chothia/Kabat 72 MAB20- L1- RASQSVSSNLA IgG4Chothia/Kabat 72 MAB21- L1- RASQSVSSNLA IgG4 Chothia/Kabat 73 SEC1Human IgG4 EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHW S228P HeavyVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRD ChainNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 74 SEC1 Heavy Chain EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVariable Region VRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDS WGQGTLVTVSS 75 SEC1 SEC1 HumanDIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKE Kappa ChainNLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 76 SEC1 Light Chain DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKEVariable Region NLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTK LEIK 77 SEC1 Mouse IgG2aEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHW N297A HeavyVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRD ChainNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDS WGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQT HREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVL DSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 74 SEC1 Heavy Chain EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVariable region VRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDS WGQGTLVTVSS 78 SEC1 MouseDIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKE Kappa ChainNLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE C 76 SEC1 Light ChainDIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKE Variable RegionNLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTK LEIK 79 MAB1 Full length IgG4QLQLQESGPGLVKPSETLSLTCTVSGGSITSSSYYWG S228PWIRQPPGKGLEWIGSIYYSGATFYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGSAWAFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 80 MAB1Full length IgG1 QLQLQESGPGLVKPSETLSLTCTVSGGSITSSSYYWGWIRQPPGKGLEWIGSIYYSGATFYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGSAWAFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 81 MAB1 Full lengthEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ KappaQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHFNLPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC 82MAB2 Full length IgG4 QLQLQESGPGLVKPSETLSLTCTVSGGSISSSKYYWG S228PWIRQPPGKGLEWIGSIYYSGSTFYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGSAWAFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK 83 MAB2Full length IgG1 QLQLQESGPGLVKPSETLSLTCTVSGGSISSSKYYWGWIRQPPGKGLEWIGSIYYSGSTFYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGSAWAF DPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 81 MAB2 Full lengthEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ KappaQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHFNLPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC 84MAB3 Full length IgG4 QLQLQESGPGLVKPSETLSLTCTVSGGSISSTSHYWG S228PWIRQPPGKGLEWIGSIYYSGSTFYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGSAWAFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSMHEALHNHYTQKSLSLSLGK 85 MAB3 Full length IgG1QLQLQESGPGLVKPSETLSLTCTVSGGSISSTSHYWGWIRQPPGKGLEWIGSIYYSGSTFYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGSAWAF DPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 81 MAB3 Full lengthEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ KappaQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHFNLPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC 86MAB4 Full length IgG4 QLQLQESGPGLVKPSETLSLTCTVSGGSISSTSHYWG S228PWIRQPPGKGLEWIGSIYYSGSTFYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGGAWAFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK 87 MAB4Full length IgG1 QLQLQESGPGLVKPSETLSLTCTVSGGSISSTSHYWGWIRQPPGKGLEWIGSIYYSGSTFYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGGAWAF DPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 81 MAB4 Full lengthEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ KappaQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHFNLPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC 88MAB5 Full length IgG4 QLQLQESGPGLVKPSETLSLTCTVSGGSISSTSHYWG S228PWIRQPPGKGLEWIGSIYYSGSTFYNPSLKGRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGSAWAFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNVVYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 89 MAB5Full length IgG1 QLQLQESGPGLVKPSETLSLTCTVSGGSISSTSHYWGWIRQPPGKGLEWIGSIYYSGSTFYNPSLKGRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDANYYGSAWAFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 81 MAB5 Full lengthEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ KappaQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHFNLPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC 90MAB6 Full length IgG4 QLQLQESGPGLVKPSETLSLTCTVSGGSIESGSYYWG S228PWIRQPPGKGLEWIGSIYYSGGTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLTLNKRSFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK 91 MAB6Full length IgG1 QLQLQESGPGLVKPSETLSLTCTVSGGSIESGSYYWGWIRQPPGKGLEWIGSIYYSGGTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLTLNKRSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 92 MAB6 Full lengthEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY KappaQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 93MAB7 Full length IgG4 QVQLQESGPGLVKPSQTLSLTCTVSGGSIESGVYYW S228PGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLTLNKRSFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 94 MAB7Full length IgG1 QVQLQESGPGLVKPSQTLSLTCTVSGGSIESGVYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLTLNKRSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 92 MAB7 Full lengthEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY KappaQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 95MAB8 Full length IgG4 QLQLQESGPGLVKPSETLSLTCTVSGGSIASGSYYWG S228PWIRQPPGKGLEWIGSIYYSGQTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLTLNKRSFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK 96 MAB8Full length IgG1 QLQLQESGPGLVKPSETLSLTCTVSGGSIASGSYYWGWIRQPPGKGLEWIGSIYYSGQTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLTLNKRSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 92 MAB8 Full lengthEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY KappaQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 97MAB9 Full length IgG4 QVQLQESGPGLVKPSQTLSLTCTVSGGSIESGLYYW S228PGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLTLNKRSFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 98 MAB9Full length IgG1 QVQLQESGPGLVKPSQTLSLTCTVSGGSIESGLYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLTLNKRSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 92 MAB9 Full lengthEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY KappaQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 99MAB10 Full length IgG4 QVQLQESGPGLVKPSQTLSLTCTVSGGSIESGLYYW S228PGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLALNKRSFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 100 MAB10Full length IgG1 QVQLQESGPGLVKPSQTLSLTCTVSGGSIESGLYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLALNKRSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 92 MAB10 Full lengthEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY KappaQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC101 MAB11 Full length IgG4 QVQLQESGPGLVKPSQTLSLTCTVSGGSIESGLYYW S228PGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLALNKRSFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 102 MAB11Full length IgG1 QVQLQESGPGLVKPSQTLSLTCTVSGGSIESGLYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLALNKRSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 92 MAB11 Full lengthEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY KappaQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC103 MAB12 Full length IgG4 QVQLQESGPGLVKPSQTLSLTCTASGGSIESGLYYW S228PGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLALNKRSFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 104 MAB12Full length IgG1 QVQLQESGPGLVKPSQTLSLTCTASGGSIESGLYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGVLALNKRSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 92 MAB12 Full lengthEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY KappaQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHTVRPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC105 MAB13 Full length IgG4 QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYYM S228PHWVRQAPGQGLEWMGIINPSLGLTSYAQKFQGRVT MTRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 106 MAB13Full length IgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYYMHWVRQAPGQGLEWMGIINPSLGLTSYAQKFQGRVT MTRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 107 MAB13Full length EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY KappaQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYVVWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC108 MAB14 Full length IgG4 QVQLVQSGAEVKKPGASVKVSCKASGYTFPAYYMH S228PWVRQAPGQGLEWMGIINPSLGLTSYAQKFQGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 109 MAB14Full length IgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFPAYYMHWVRQAPGQGLEWMGIINPSLGLTSYAQKFQGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 107 MAB14 Full lengthEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY KappaQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYVVWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC110 MAB15 Full length IgG4 QVQLVQSGAEVKKPGASVKVSCKASGYTFREYYMH S228PWVRQAPGQGLEWMGIINPSIGLTSYARKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 111 MAB15Full length IgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFREYYMHWVRQAPGQGLEWMGIINPSIGLTSYARKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 107 MAB15 Full lengthEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY KappaQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYVVWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC112 MAB16 Full length IgG4 QVQLVQSGAEVKKPGASVKVSCKASGYTFREYYMH S228PWVRQAPGQGLEWMGIINPSIGLTSYARKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGALDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 113 MAB16Full length IgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFREYYMHWVRQAPGQGLEWMGIINPSIGLTSYARKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGALDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 107 MAB16 Full lengthEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY KappaQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYVVWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC114 MAB17 Full length IgG4 QVQLVQSGAEVKKPGASVKVSCKASGYTFPAYYIH S228PWVRQAPGQGLEWMGIINPSLGLTSYARKFQGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGALDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 115 MAB17Full length IgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFPAYYIHWVRQAPGQGLEWMGIINPSLGLTSYARKFQGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGALDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 107 MAB17 Full lengthEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY KappaQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYVVWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC116 MAB18 Full length IgG4 QVQLVQSGAEVKKPGASVKVSCKASGYTFPAYYMH S228PWVRQAPGQGLEWMGIINPSLGLTSYARKFQGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 117 MAB18Full length IgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFPAYYMHWVRQAPGQGLEWMGIINPSLGLTSYARKFQGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCARGGRTTWIGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 107 MAB18 Full lengthEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY KappaQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYVVWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC118 MAB19 Full length IgG4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHYMG S228PWVRQAPGQGLEWMGVINPSMGATSYAQKFQGRVT MTRDTSTSTVYMELSSLRSEDTAVYYCARLHVSGSYYPAYLDYWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK 119 MAB19Full length IgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHYMGWVRQAPGQGLEWMGVINPSMGATSYAQKFQGRVT MTRDTSTSTVYMELSSLRSEDTAVYYCARLHVSGSYYPAYLDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 120 MAB19Full length EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY KappaQQKPGQAPRHLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYIVFPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 121MAB20 Full length IgG4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHYMG S228PWVRQAPGQGLEWVGIINPSMGATSYAQKFQGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCARLHVSGSYYPAYLDYWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 122 MAB20 Full length IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHYMG WVRQAPGQGLEWVGIINPSMGATSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLHVSGSYYPAYLDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 120 MAB20Full length EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY KappaQQKPGQAPRHLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYIVFPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 123MAB21 Full length IgG4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHYMG S228PWVRQAPGQGLEWMGIINPSMGATSYTQKFRGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCARLHVSGSYYPAYLDYWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 124 MAB21 Full length IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHYMG WVRQAPGQGLEWMGIINPSMGATSYTQKFRGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLHVSGSYYPAYLDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 120 MAB21Full length EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY KappaQQKPGQAPRHLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYIVFPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 125IgG1 Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV (G1m(17,1)TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS allotype, N297ASLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 126 Kappa ConstantRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC127 Linker GGGGS128 . . . 137 - See other portions of this disclosure and the electronic versionof the Sequence Listing submitted herewith. 138 mTIGIT2MHGWLLLVWVQGLIQAAFLATAIGATAGTIDTKRNISAEEGGSVILQCHFSSDTAEVTQVDWKQQDQLLAIYSVDLGWHVASVFSDRVVPGPSLGLTFQSLTMNDTGEYFCTYHTYPGGIYKGRIFLKVQESSDDRNGLAQFQTAPLGGTMAAVLGLICLMVTGVTVLARKDKSIRMHSIESGLGRTEAEPQEWNLRSLSSPGSPVQTQTAPAGPC GEQAEDDYADPQEYFNVLSYRSLESFIAVSKTG

What is claimed is:
 1. A method of enhancing an immune response in ahuman subject in need thereof, comprising administering to the subjectan effective amount of an isolated antibody that specifically bindshuman TIGIT (hTIGIT; SEQ ID NO: 1), comprising a heavy chain variable(VH) domain comprising from N-terminus to C-terminus, CDR-H1, CDR-H2,and CDR-H3; and a light chain variable (VL) domain comprising fromN-terminus to C-terminus CDR-L1, CDR-L2, and CDR-L3, wherein the CDRs ofthe VH and VL comprise the following six CDR sequences: (a) a CDR-H3having the sequence A-R-D-G-V-L-X₁-L-N-K-R-S-F-D-I, wherein X₁ is A or T(SEQ ID NO: 128); (b) a CDR-H2 having the sequenceS-I-Y-Y-S-G-X₂-T-Y-Y-N-P-S-L-K-S, wherein X₂ is S, Q or G (SEQ ID NO:129); (c) a CDR-H1 having the sequence G-S-I-X₃-S-G-X₄-Y-Y-W-G, whereinX₃ is E or A, and X₄ is L, V or S (SEQ ID NO: 130); (d) a CDR-L3 havingthe sequence QQHTVRPPLT (SEQ ID NO: 64); (e) a CDR-L2 having thesequence GASSRAT (SEQ ID NO: 68); and (f) a CDR-L1 having the sequenceRASQSVSSSYLA (SEQ ID NO: 71), or a pharmaceutical composition thereof,and further comprising administering at least one additional therapeuticagent.
 2. The method of claim 1, wherein the antibody comprises: (a) aCDR-H3 of SEQ ID NO: 32, a CDR-H2 of SEQ ID NO: 40, a CDR-H1 of SEQ IDNO: 54, a CDR-L3 of SEQ ID NO: 64, a CDR-L2 of SEQ ID NO: 68, and aCDR-L1 of SEQ ID NO: 71; (b) a CDR-H3 of SEQ ID NO: 31, a CDR-H2 of SEQID NO: 40, a CDR-H1 of SEQ ID NO: 54, a CDR-L3 of SEQ ID NO: 64, aCDR-L2 of SEQ ID NO: 68, and a CDR-L1 of SEQ ID NO: 71; (c) a CDR-H3 ofSEQ ID NO: 31, a CDR-H2 of SEQ ID NO: 39, a CDR-H1 of SEQ ID NO: 51, aCDR-L3 of SEQ ID NO: 64, a CDR-L2 of SEQ ID NO: 68, and a CDR-L1 of SEQID NO: 71; (d) a CDR-H3 of SEQ ID NO: 31, a CDR-H2 of SEQ ID NO: 40, aCDR-H1 of SEQ ID NO: 52, a CDR-L3 of SEQ ID NO: 64, a CDR-L2 of SEQ IDNO: 68, and a CDR-L1 of SEQ ID NO: 71; or (e) a CDR-H3 of SEQ ID NO: 31,a CDR-H2 of SEQ ID NO: 41, a CDR-H1 of SEQ ID NO: 53, a CDR-L3 of SEQ IDNO: 64, a CDR-L2 of SEQ ID NO: 68, and a CDR-L1 of SEQ ID NO:
 71. 3. Themethod of claim 1, wherein the antibody that specifically binds humanTIGIT is MAB6, MAB7, MAB8, MAB9, MAB10, MAB11, or MAB12, wherein MAB6comprises a VH domain of SEQ ID NO: 9, and a VL domain of SEQ ID NO: 26;MAB7 comprises a VH domain of SEQ ID NO: 10, and a VL domain of SEQ IDNO: 26; MAB8 comprises a VH domain of SEQ ID NO: 11, and a VL domain ofSEQ ID NO: 26; MAB9 comprises a VH domain of SEQ ID NO: 12, and a VLdomain of SEQ ID NO: 26; MAB10 comprises a VH domain of SEQ ID NO: 13,and a VL domain of SEQ ID NO: 26: MAB11 comprises a VH domain of SEQ IDNO: 14, and a VL domain of SEQ ID NO: 26; and MAB12 comprises a VHdomain of SEQ ID NO: 15, and a VL domain of SEQ ID NO:
 26. 4. The methodof claim 1, wherein the antibody that specifically binds human TIGIT isMAB10, wherein MAB10 comprises a VH domain of SEQ ID NO: 13, and a VLdomain of SEQ ID NO:
 26. 5. The method of claim 1, wherein the at leastone additional therapeutic agent is an agent that inhibits theinteraction between PD-1 and PD-L, and wherein the agent that inhibitsthe interaction between PD-1 and PD-L1 is selected from an antibody, apeptidomimetic, or a small molecule.
 6. The method of claim 5, whereinthe agent that inhibits the interaction between PD-1 and PD-L1 isselected from the group consisting of pembrolizumab, nivolumab,atezolizumab, avelumab, durvalumab and BMS-936559.
 7. The method ofclaim 1, wherein the subject is a subject that has been treated with anagent that inhibits the interaction between PD-1 and PD-L1 prior toperforming such method.
 8. The method of claim 1, wherein the subjectwas not responsive to a prior therapy with the at least one additionaltherapeutic agent.
 9. The method of claim 1, wherein the additionaltherapeutic agent is an immunostimulatory agent selected from (a) anagent that blocks signaling of an inhibitory receptor of an immune cellor a ligand thereof; (b) an agonist to a stimulatory receptor of animmune cell; (c) a cytokine; (d) an oncolytic virus; (e) a T cellexpressing a chimeric antigen receptor; (f) a bi- or multi-specific Tcell directed antibody; (g) an anti-TGF-β antibody; (h) a TGF-β trap;(i) a vaccine to a cancer-associated antigen, including such antigen ora nucleic acid encoding such antigen and (j) combinations thereof. 10.The method of claim 9, wherein the additional therapeutic agent is anagent that blocks signaling of an inhibitory receptor of an immune cellor a ligand thereof, and the inhibitory receptor or ligand thereof isselected from CTLA-4, PD-L2, LAG-3, Tim3, neuritin, BTLA, CECAM-1,CECAM-5, VISTA, LAIR1, CD160, 2B4, TGF-R, KIR, and combinations thereof.11. The method of claim 9, wherein the additional therapeutic agent isan agonist to a stimulatory receptor of an immune cell, and thestimulatory receptor of an immune cell is selected from OX40, CD2, CD27,CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR,CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160,B7-H3, CD83 ligand, and combinations thereof.
 12. A method of treating acancer in a human subject in need thereof, comprising administering tothe subject an effective amount of an isolated antibody thatspecifically binds human TIGIT (hTIGIT; SEQ ID NO: 1), comprising aheavy chain variable (VH) domain comprising from N-terminus toC-terminus, CDR-H1, CDR-H2, and CDR-H3; and a light chain variable (VL)domain comprising from N-terminus to C-terminus CDR-L1, CDR-L2, andCDR-L3, wherein the CDRs of the VH and VL comprise the following six CDRsequences: (a) a CDR-H3 having the sequenceA-R-D-G-V-L-X₁-L-N-K-R-S-F-D-I, wherein X₁ is A or T (SEQ ID NO: 128);(b) a CDR-H2 having the sequence S-I-Y-Y-S-G-X₂-T-Y-Y-N-P-S-L-K-S,wherein X₂ is S, Q or G (SEQ ID NO: 129); (c) a CDR-H1 having thesequence G-S-I-X₃-S-G-X₄-Y-Y-W-G, wherein X₃ is E or A, and X₄ is L, Vor S (SEQ ID NO: 130); (d) a CDR-L3 having the sequence QQHTVRPPLT (SEQID NO: 64); (e) a CDR-L2 having the sequence GASSRAT (SEQ ID NO: 68);and (f) a CDR-L1 having the sequence RASQSVSSSYLA (SEQ ID NO: 71); andfurther comprising administering at least one additional therapeuticagent selected from an immunomodulatory agent, a chemotherapeutic agent,an immunostimulatory agent, radiation, and combinations thereof.
 13. Themethod of claim 12, wherein the immunomodulatory agent comprises anagent that inhibits the interaction between PD-1 and PD-L1, and whereinthe agent that inhibits the interaction between PD-1 and PD-L1 isselected from an antibody, a peptidomimetic, or a small molecule. 14.The method of claim 13, wherein the agent that inhibits the interactionbetween PD-1 and PD-L1 is selected from the group consisting ofpembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, andBMS-936559.
 15. The method of claim 12, wherein the antibody thatspecifically binds human TIGIT comprises: (a) a CDR-H3 of SEQ ID NO: 32,a CDR-H2 of SEQ ID NO: 40, a CDR-H1 of SEQ ID NO: 54, a CDR-L3 of SEQ IDNO: 64, a CDR-L2 of SEQ ID NO: 68, and a CDR-L1 of SEQ ID NO: 71; (b) aCDR-H3 of SEQ ID NO: 31, a CDR-H2 of SEQ ID NO: 40, a CDR-H1 of SEQ IDNO: 54, a CDR-L3 of SEQ ID NO: 64, a CDR-L2 of SEQ ID NO: 68, and aCDR-L1 of SEQ ID NO: 71; (c) a CDR-H3 of SEQ ID NO: 31, a CDR-H2 of SEQID NO: 39, a CDR-H1 of SEQ ID NO: 51, a CDR-L3 of SEQ ID NO: 64, aCDR-L2 of SEQ ID NO: 68, and a CDR-L1 of SEQ ID NO: 71; (d) a CDR-H3 ofSEQ ID NO: 31, a CDR-H2 of SEQ ID NO: 40, a CDR-H1 of SEQ ID NO: 52, aCDR-L3 of SEQ ID NO: 64, a CDR-L2 of SEQ ID NO: 68, and a CDR-L1 of SEQID NO: 71; or (e) a CDR-H3 of SEQ ID NO: 31, a CDR-H2 of SEQ ID NO: 41,a CDR-H1 of SEQ ID NO: 53, a CDR-L3 of SEQ ID NO: 64, a CDR-L2 of SEQ IDNO: 68, and a CDR-L1 of SEQ ID NO:
 71. 16. The method of claim 12,wherein the antibody that specifically binds human TIGIT is MAB6, MAB7,MAB8, MAB9, MAB10, MAB11, OR MAB12, wherein MAB6 comprises a VH domainof SEQ ID NO: 9, and a VL domain of SEQ ID NO: 26; MAB7 comprises a VHdomain of SEQ ID NO: 10, and a VL domain of SEQ ID NO: 26; MAB8comprises a VH domain of SEQ ID NO: 11, and a VL domain of SEQ ID NO:26; MAB9 comprises a VH domain of SEQ ID NO: 12, and a VL domain of SEQID NO: 26; MAB10 comprises a VH domain of SEQ ID NO: 13, and a VL domainof SEQ ID NO: 26: MAB11 comprises a VH domain of SEQ ID NO: 14, and a VLdomain of SEQ ID NO: 26; and MAB12 comprises a VH domain of SEQ ID NO:15, and a VL domain of SEQ ID NO:
 26. 17. The method of claim 12,wherein the antibody that specifically binds human TIGIT is MAB10,wherein MAB10 comprises a VH domain of SEQ ID NO: 13, and a VL domain ofSEQ ID NO:
 26. 18. The method of claim 12, wherein the subject is asubject that has been treated with an agent that inhibits theinteraction between PD-1 and PD-L1 prior to performing such method. 19.The method of claim 12, wherein the cancer in the subject was notresponsive to a prior therapy with the at least one additionaltherapeutic agent.
 20. The method of claim 12, wherein at least oneadditional therapeutic agent is an agent selected from achemotherapeutic agent, an immunostimulatory agent, radiation, andcombinations thereof.
 21. The method of claim 12, wherein the antibodythat specifically binds human TIGIT is MAB10, wherein MAB10 comprises aVH domain of SEQ ID NO: 13, and a VL domain of SEQ ID NO: 26, andwherein the immunomodulatory agent comprises an agent that inhibits theinteraction between PD-1 and PD-L1 selected from the group consisting ofan antibody, a peptidomimetic, and a small molecule.
 22. The method ofclaim 21, wherein the agent that inhibits the interaction between PD-1and PD-L1 is selected from the group consisting of pembrolizumab,nivolumab, atezolizumab, avelumab, durvalumab, and BMS-936559.
 23. Themethod of claim 22, wherein the agent that inhibits the interactionbetween PD1 and PD-L1 is pembrolizumab.
 24. The method of claim 22,wherein the agent that inhibits the interaction between PD1 and PD-L1 isnivolumab.